Anti-alk2 antibody

ABSTRACT

This application provides: an antibody which specifically binds to an ALK2 protein and has an activity of inhibiting BMP signal transduction mediated by ALK2; a method for producing the antibody; and a pharmaceutical composition comprising the antibody, for treating and/or preventing ectopic ossification and/or bone dysplasia, anemia, or diffuse intrinsic pontine glioma (DIPG).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.15/547,231, which is the U.S. National Stage application ofPCT/JP2016/052602, filed Jan. 29, 2016, which claims priority fromJapanese application JP 2015-017882, filed Jan. 30, 2015.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 19, 2019, isnamed sequence.txt and is 204,800 bytes.

TECHNICAL FIELD

The present invention relates to a substance useful as a therapeuticand/or prophylactic drug for ectopic ossification and/or bone dysplasia,and to a method for treating and/or preventing ectopic ossificationand/or bone dysplasia.

BACKGROUND ART

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease whichcauses cartilage tissues or bone tissues to be ectopically formed insoft tissues, such as skeletal muscle, tendon, and ligament, where bonetissues are not normally formed (Non Patent Literatures 1 to 3). In thisdisease, ectopic ossification occurs throughout the entire bodyincluding the face so that ectopic bones and existing normal bones arefused to remarkably reduce the range of joint motion or to deform thebody (Non Patent Literatures 1 to 3).

The ectopic ossification in FOP proceeds chronically with growth,whereas acute ectopic ossification is also known which proceeds whilemanifesting a symptom called flare-up caused by muscle injury, viralinfection, or the like (Non Patent Literature 1). The flare-up isswelling with inflammatory response or sustained pain as principalsymptoms. The flare-up is induced by bruise, falling, intramuscularinjection, or the like, which causes muscle injury. In addition, suddenflare-ups with no clear cause are also known. For FOP, invasive medicalacts such as biopsy and operation are contraindicated because ectopicbones are formed after flare-up. Thus, the ectopic bone tissues cannotbe surgically removed. The ectopic bone tissues in FOP are consisted ofnormal cartilage cells or osteoblasts and bone metabolismee ofheterotopic bones is the same as normal bone tissues. As such, only theectopic bone tissues cannot be medically removed using drugs or thelike.

Any fundamental therapy for suppressing the ectopic ossification in FOPhas not yet been established, and only symptomatic treatment for pain orthe like has been made. Thus, the ectopic bone tissues formed in FOP arevery difficult to remove, and the development of a promising drug thatcan exert prophylactic effects before the onset of ectopic ossificationhas been expected.

Activin like kinase 2 (ALK2) gene encoding a receptor of bonemorphogenetic proteins (BMPs) that induces ectopic bone formation insoft tissues including skeletal muscle tissues has been identified as aresponsible gene for FOP (Non Patent Literature 4). The ALK2 gene isidentical to activin A type I receptor 1 (ACVR1) gene. ALK2 having anamino acid substitution has been found from familial and sporadic FOPcases (Non Patent Literature 4).

Human or mouse ALK2 is a single-pass transmembrane protein consisting of509 amino acids and having a signal peptide and functions as atransmembrane type of serine-threonine kinase receptor binding to BMPs(Non Patent Literatures 1 to 3). ALK2 binds to BMPs at its N-terminalextracellular region and activates the downstream intracellular signaltransduction system through an intracellular serine-threonine kinase.

BMP receptors are classified based on their structures and functionsinto 2 types: type I receptors including ALK2; and type II receptors(Non Patent Literatures 1 to 3). The type II receptors areconstitutively active enzymes that exhibit kinase activity even if notbound with BMP. On the other hand, the type I receptors including ALK2are inactive enzymes in a state unbound with BMP and exhibit kinaseactivity in a manner dependent on binding to BMP. This is probablybecause upon binding to BMP, type II receptor kinase phosphorylates typeI receptor intracellular domain as the substrate, which can change itsconformation, and activates the type I receptor (Non Patent Literatures1 to 3).

Type I receptors are known to be constitutive active independent of atype II receptor by substitution of a particular amino acid in theintracellular region (Non Patent Literatures 1 to 3). Overexpression ofthis constitutively active mutants of the type I receptors activate theintracellular signal transduction system without BMP stimulation. Thus,the type I receptors are considered as responsible molecules thattransduce BMP signals from the outside to the inside of cells.

A mutation in ALK2 identified from familial and typical sporadic FOPpatients was the R206H mutation in which Arg206 is substituted by His(Non Patent Literature 4). All of gene mutations previously identifiedfrom FOP cases have been reported to cause amino acid substitutions inthe intracellular region of ALK2. Most of these mutations from FOP casesreside mainly in or around the ATP-binding region in the intracellulardomain of ALK2 (Non Patent Literature 5).

Overexpression of the ALK2 mutants identified in FOP in cultured cellsactivates the intracellular signal transduction system of BMP withoutBMP stimulation (Non Patent Literature 6). Accordingly, for example,small molecular inhibitors for ALK2 kinase, RNAi or exon skippingmethods which specifically suppress the expression of geneticallymutated ALK2, downstream transcriptional factor inhibitors of ALK2receptor, and inhibitors of BMP signal-mediated osteoblastdifferentiation are being developed as therapeutics for FOP (PatentLiterature 1 and Non Patent Literatures 1 to 3).

All of small molecular compounds or nucleic acids currently underdevelopment as therapeutics for FOP are expected to inhibitintracellular ALK2 signal after they penetrate cell membranes. However,any effective drug delivery method has not yet been established fornucleic acid drugs. In addition, there still remain problems such as thelow specificity of kinase inhibitors for other ALK receptor familieswith high similarity to ALK2. As such, it has been desired to developnovel therapeutics for FOP having high specificity for ALK2. Antibodydrugs that act on the extracellular region of ALK2 and inhibit itssignal are highly safe therapeutic approach that utilize thephysiological immune system. The antibody drugs facilitate stable drugdelivery via blood flow and are capable of specifically inhibiting ALK2without acting on other ALK receptor families with high identitythereto. Furthermore, the antibody drugs can be expected to haveinhibitory effect on wild-type ALK2 and various intracellular mutantALK2s including novel unidentified mutants. The antibody drugs areexpected not to inhibit ALK3 expressed in cells throughout the body.Therefore, unlike general osteoblast differentiation inhibitors,antibodies specifically inhibiting ALK2 can be expected to serve asdrugs less likely to influence the growth, maintenance, and regenerationof normal skeletal tissues. However, any antibody that specificallybinds to ALK2 and exhibits therapeutic effects on FOP has not been knownso far.

PRIOR ART LITERATURE Patent Literature

-   Patent Literature 1: International Publication No. WO2007/123896

Non Patent Literature

-   Non Patent Literature 1: T. Katagiri, J. Oral Biosci., 52, 33-41    (2010)-   Non Patent Literature 2: T. Katagiri, J. Oral Biosci., 54, 119-123    (2012)-   Non Patent Literature 3: T. Katagiri and S. Tsukamoto, Biol. Chem.,    394, 703-714 (2013)-   Non Patent Literature 4: E. M. Shore et al., Nat. Genet., 38,    525-527 (2006)-   Non Patent Literature 5: A. Chaikuad et al., J. Biol. Chem., 287,    36990-36998 (2012)-   Non Patent Literature 6: T. Fukuda et al., J. Biol. Chem., 284,    7149-7156 (2009)

SUMMARY OF INVENTION Problem to be Solved by the Invention

An object of the present invention is to provide a substance useful as atherapeutic and/or prophylactic drug for ectopic ossification and/orbone dysplasia, and a method for treating and/or preventing ectopicossification and/or bone dysplasia.

Means for Solution of the Problem

The present inventors have now conducted diligent studies to attain theobject and consequently completed the present invention throughsuccessfully obtaining a novel antibody that specifically binds to ALK2and has therapeutic and/or prophylactic effects on ectopic ossificationand/or bone dysplasia.

Specifically, the present invention encompasses the following aspects:

(1) An antibody or an antigen-binding fragment thereof whichspecifically binds to a polypeptide sequence consisting of at least 7amino acids in any one of the following amino acid sequences (a) to (e)and inhibits ALK2-mediated BMP signal transduction:

(a) the amino acid sequence of SEQ ID NO: 84;(b) an amino acid sequence consisting of amino acid numbers 21 to 123 ofthe amino acid sequence of SEQ ID NO: 84;(c) the amino acid sequence of SEQ ID NO: 86;(d) an amino acid sequence consisting of amino acid numbers 21 to 123 ofthe amino acid sequence of SEQ ID NO: 86; and(e) an amino acid sequence comprising a substitution(s), deletion(s), oraddition(s) of one to several amino acid residues in any one of theamino acid sequences (a) to (d).

(2) An antibody or an antigen-binding fragment thereof whichspecifically binds to a polypeptide sequence consisting of at least 7amino acids in an amino acid sequence encoded by any one of thefollowing nucleotide sequences (f) to (j) and inhibits ALK2-mediated BMPsignal transduction:

(f) the nucleotide sequence of SEQ ID NO: 85;(g) a nucleotide sequence consisting of nucleotide numbers 728 to 1036of the nucleotide sequence of SEQ ID NO: 85;(h) the nucleotide sequence of SEQ ID NO: 87;(i) a nucleotide sequence consisting of nucleotide numbers 728 to 1036of the nucleotide sequence of SEQ ID NO: 87; and(j) a nucleotide sequence of a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of the nucleotidesequence complementary to any one of the nucleotide sequences (f) to(i).

(3) The antibody or the antigen-binding fragment thereof according to(1) or (2), wherein the polypeptide sequence is a sequence in an ALK2extracellular region.

(4) The antibody or the antigen-binding fragment thereof according toany one of (1) to (3), wherein the antibody binds to a wild-type ALK2protein and a mutant ALK2 protein.

(5) The antibody or the antigen-binding fragment thereof according toany one of (1) to (4), wherein the antibody is a monoclonal antibody ora polyclonal antibody.

(6) The antibody or the antigen-binding fragment thereof according toany one of (1) to (5), wherein the antibody or the antigen-bindingfragment is a chimeric antibody, a humanized antibody, a human antibody,a single-chain antibody, a bispecific antibody, or a multispecificantibody.

(7) The antibody or the antigen-binding fragment thereof according toany one of (1) to (6), wherein the antibody or the antigen-bindingfragment cross-competes, for binding to the polypeptide, with at leastany one antibody selected from the group consisting of an antibodycomprising a heavy chain variable region consisting of the amino acidsequence of SEQ ID NO: 2 and a light chain variable region consisting ofthe amino acid sequence of SEQ ID NO: 4, an antibody comprising a heavychain variable region consisting of the amino acid sequence of SEQ IDNO: 6 and a light chain variable region consisting of the amino acidsequence of SEQ ID NO: 8, an antibody comprising a heavy chain variableregion consisting of the amino acid sequence of SEQ ID NO: 10 and alight chain variable region consisting of the amino acid sequence of SEQID NO: 12, and an antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of SEQ ID NO: 14 and a light chainvariable region consisting of the amino acid sequence of SEQ ID NO: 16.

(8) The antibody or the antigen-binding fragment thereof according toany one of (1) to (6), wherein the antibody or the antigen-bindingfragment binds to an epitope that is bound by at least any one antibodyselected from the group consisting of an antibody comprising a heavychain variable region consisting of the amino acid sequence of SEQ IDNO: 2 and a light chain variable region consisting of the amino acidsequence of SEQ ID NO: 4, an antibody comprising a heavy chain variableregion consisting of the amino acid sequence of SEQ ID NO: 6 and a lightchain variable region consisting of the amino acid sequence of SEQ IDNO: 8, an antibody comprising a heavy chain variable region consistingof the amino acid sequence of SEQ ID NO: 10 and a light chain variableregion consisting of the amino acid sequence of SEQ ID NO: 12, and anantibody comprising a heavy chain variable region consisting of theamino acid sequence of SEQ ID NO: 14 and a light chain variable regionconsisting of the amino acid sequence of SEQ ID NO: 16.

(9) The antibody or the antigen-binding fragment thereof according toany one of (1) to (6), wherein the antibody or the antigen-bindingfragment binds to an epitope comprising each residue of glutamic acid(Glu) at position 18, glycine (Gly) at position 19, isoleucine (Ile) atposition 39, asparagine (Asn) at position 40, aspartic acid (Asp) atposition 41, glycine (Gly) at position 42, phenylalanine (Phe) atposition 43, histidine (His) at position 44, valine (Val) at position45, tyrosine (Tyr) at position 46, asparagine (Asn) at position 82,threonine (Thr) at position 84, glutamine (Gln) at position 86, leucine(Leu) at position 87, proline (Pro) at position 88, and threonine (Thr)at position 89 in the amino acid sequence of SEQ ID NO: 84.

(10) The antibody or the antigen-binding fragment thereof according toany one of (1) to (6), having an interaction distance between theantibody or the antigen-binding fragment and each residue of glutamicacid (Glu) at position 18, glycine (Gly) at position 19, isoleucine(Ile) at position 39, asparagine (Asn) at position 40, aspartic acid(Asp) at position 41, glycine (Gly) at position 42, phenylalanine (Phe)at position 43, histidine (His) at position 44, valine (Val) at position45, tyrosine (Tyr) at position 46, asparagine (Asn) at position 82,threonine (Thr) at position 84, glutamine (Gln) at position 86, leucine(Leu) at position 87, proline (Pro) at position 88, and threonine (Thr)at position 89 in the amino acid sequence of SEQ ID NO: 84.

(11) The antibody or the antigen-binding fragment thereof according toany one of (1) to (6), wherein the antibody or the antigen-bindingfragment binds to an epitope comprising each residue of glutamic acid(Glu) at position 18, glycine (Gly) at position 19, leucine (Leu) atposition 20, isoleucine (Ile) at position 39, asparagine (Asn) atposition 40, aspartic acid (Asp) at position 41, glycine (Gly) atposition 42, phenylalanine (Phe) at position 43, histidine (His) atposition 44, valine (Val) at position 45, tyrosine (Tyr) at position 46,and threonine (Thr) at position 84 in the amino acid sequence of SEQ IDNO: 84.

(12) The antibody or the antigen-binding fragment thereof according toany one of (1) to (6), having an interaction distance between theantibody or the antigen-binding fragment and each residue of glutamicacid (Glu) at position 18, glycine (Gly) at position 19, leucine (Leu)at position 20, isoleucine (Ile) at position 39, asparagine (Asn) atposition 40, aspartic acid (Asp) at position 41, glycine (Gly) atposition 42, phenylalanine (Phe) at position 43, histidine (His) atposition 44, valine (Val) at position 45, tyrosine (Tyr) at position 46,and threonine (Thr) at position 84 in the amino acid sequence of SEQ IDNO: 84.

(13) The antibody or the antigen-binding fragment thereof according to(10) or (12), wherein the interaction distance is 6 angstroms orsmaller.

(14) The antibody or the antigen-binding fragment thereof according to(10) or (12), wherein the interaction distance is 4 angstroms orsmaller.

(15) The antibody or the antigen-binding fragment thereof according toany one of (1) to (8), wherein

the heavy chain sequence comprises a variable region having CDRH1,CDRH2, and CDRH3, wherein the CDRH1 consists of the amino acid sequenceof SEQ ID NO: 72, the CDRH2 consists of the amino acid sequence of SEQID NO: 73, and the CDRH3 consists of the amino acid sequence of SEQ IDNO: 74, andthe light chain sequence comprises a variable region having CDRL1,CDRL2, and CDRL3, wherein the CDRL1 consists of the amino acid sequenceof SEQ ID NO: 75, the CDRL2 consists of the amino acid sequence of SEQID NO: 76, and the CDRL3 consists of the amino acid sequence of SEQ IDNO: 77.

(16) The antibody or the antigen-binding fragment thereof according to(15), wherein the antibody comprises a heavy chain variable regionsequence consisting of the amino acid sequence of SEQ ID NO: 2 and alight chain variable region sequence consisting of the amino acidsequence of SEQ ID NO: 4.

(17) The antibody or the antigen-binding fragment thereof according toany one of (1) to (8), wherein

the heavy chain sequence comprises a variable region having CDRH1,CDRH2, and CDRH3, wherein the CDRH1 consists of an amino acid sequenceof SEQ ID NO: 59, the CDRH2 consists of an amino acid sequence of SEQ IDNO: 60, and the CDRH3 consists of an amino acid sequence of SEQ ID NO:61, andthe light chain sequence comprises a variable region having CDRL1,CDRL2, and CDRL3, wherein the CDRL1 consists of the amino acid sequenceof SEQ ID NO: 62, the CDRL2 consists of the amino acid sequence of SEQID NO: 63 or SEQ ID NO: 71, and the CDRL3 consists of the amino acidsequence of SEQ ID NO: 64.

(18) The antibody or the antigen-binding fragment thereof according to(17), wherein the antibody comprises a heavy chain variable regionsequence consisting of the amino acid sequence of SEQ ID NO: 6 and alight chain variable region sequence consisting of the amino acidsequence of SEQ ID NO: 8 or the amino acid numbers 21 to 133 of theamino acid sequence of SEQ ID NO: 36.

(19) The antibody or the antigen-binding fragment of the antibodyaccording to any one of (1) to (10), (13) and (14), wherein

a heavy chain sequence comprises a variable region having CDRH1, CDRH2,and CDRH3, wherein the CDRH1 consists of the amino acid sequence of SEQID NO: 78, the CDRH2 consists of the amino acid sequence of SEQ ID NO:79, and the CDRH3 consists of the amino acid sequence of SEQ ID NO: 80;anda light chain sequence comprises a variable region having CDRL1, CDRL2,and CDRL3, wherein the CDRL1 consists of the amino acid sequence of SEQID NO: 81, the CDRL2 consists of the amino acid sequence of SEQ ID NO:82, and the CDRL3 consists of the amino acid sequence of SEQ ID NO: 83.

(20) The antibody or the antigen-binding fragment thereof according to(19), wherein the antibody comprises a heavy chain variable regionsequence consisting of the amino acid sequence of SEQ ID NO: 10 and alight chain variable region sequence consisting of the amino acidsequence of SEQ ID NO: 12.

(21) The antibody or the antigen-binding fragment thereof according toany one of (1) to (8) and (11) to (14), wherein

a heavy chain sequence comprises a variable region having CDRH1, CDRH2,and CDRH3, wherein the CDRH1 consists of the amino acid sequence of SEQID NO: 65, the CDRH2 consists of the amino acid sequence of SEQ ID NO:66, and the CDRH3 consists of the amino acid sequence of SEQ ID NO: 67;anda light chain sequence comprises a variable region having CDRL1, CDRL2,and CDRL3, wherein the CDRL1 consists of the amino acid sequence of SEQID NO: 68, the CDRL2 consists of the amino acid sequence of SEQ ID NO:69, and the CDRL3 consists of the amino acid sequence of SEQ ID NO: 70.

(22) The antibody or the antigen-binding fragment thereof according to(21), wherein the antibody comprises a heavy chain variable regionsequence consisting of the amino acid sequence of SEQ ID NO: 14 and alight chain variable region sequence consisting of the amino acidsequence of SEQ ID NO: 16.

(23) The antigen-binding fragment thereof according to any one of (1) to(22), wherein the antigen-binding fragment is selected from the groupconsisting of Fab, F(ab′)2, Fab′, and Fv.

(24) The antibody according to any one of (1) to (22), wherein theantibody is scFv.

(25) The antibody or the antigen-binding fragment thereof according toany one of (1) to (22), wherein the antibody is a chimeric antibody.

(26) The antibody or the antigen-binding fragment thereof according toany one of (1) to (22), wherein the antibody is humanized.

(27) The antibody according to any one of (1) to (24), wherein the heavychain comprises a constant region of a human immunoglobulin G1 heavychain, a human immunoglobulin G2 heavy chain, or a human immunoglobulinG4 heavy chain, and the light chain comprises a constant region of ahuman immunoglobulin K light chain.

(28) The antibody according to (27), wherein the heavy chain comprises aconstant region of a human immunoglobulin G1 heavy chain.

(29) The antibody according to (28), wherein in the human immunoglobulinG1 heavy chain, leucine (Leu) at position 234 is substituted by alanine(Ala), and leucine (Leu) at position 235 is substituted by alanine(Ala).

(30) The antibody according to (27), wherein the heavy chain comprises aconstant region of a human immunoglobulin G2 heavy chain.

(31) The antibody according to (27), wherein the heavy chain comprises aconstant region of a human immunoglobulin G4 heavy chain.

(32) The antibody according to (31), wherein in the human immunoglobulinG4 heavy chain, serine (Ser) at position 241 is substituted by proline(Pro).

(33) An antibody or an antigen-binding fragment thereof whichspecifically binds to an extracellular region of ALK2 protein andinhibits ALK2-mediated BMP signal transduction, wherein the antibody orthe antigen-binding fragment comprises

(a) a heavy chain variable region sequence selected from the groupconsisting of the following amino acid sequences:a1) an amino acid sequence consisting of amino acid numbers 20 to 142 ofthe amino acid sequence of SEQ ID NO: 28;a2) an amino acid sequence consisting of amino acid numbers 20 to 142 ofthe amino acid sequence of SEQ ID NO: 30;a3) an amino acid sequence consisting of amino acid numbers 20 to 142 ofthe amino acid sequence of SEQ ID NO: 105;a4) an amino acid sequence having at least 95% identity to any of theamino acid sequences a1) to a3);a5) an amino acid sequence having at least 99% identity to any of theamino acid sequences a1) to a3); anda6) an amino acid sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several amino acid residues in any of theamino acid sequences a1) to a3); and(b) a light chain variable region sequence selected from the groupconsisting of the following amino acid sequences:b1) an amino acid sequence consisting of amino acid numbers 21 to 133 ofthe amino acid sequence of SEQ ID NO: 32;b2) an amino acid sequence consisting of amino acid numbers 21 to 133 ofthe amino acid sequence of SEQ ID NO: 34;b3) an amino acid sequence consisting of amino acid numbers 21 to 133 ofthe amino acid sequence of SEQ ID NO: 36;b4) an amino acid sequence consisting of amino acid numbers 21 to 133 ofthe amino acid sequence of SEQ ID NO: 38;b5) an amino acid sequence having at least 95% identity to any one aminoacid sequence selected from the amino acid sequences b1) to b4);b6) an amino acid sequence having at least 99% identity to any one aminoacid sequence selected from the amino acid sequences b1) to b4); andb7) an amino acid sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several amino acid residues in any one aminoacid sequence selected from the amino acid sequences b1) to b4).

(34) The antibody or the antigen-binding fragment thereof according to(33), wherein the antibody is an antibody consisting of a heavy chaincomprising a heavy chain variable region sequence consisting of aminoacid numbers 20 to 142 of the amino acid sequence of SEQ ID NO: 30 and alight chain comprising a light chain variable region sequence consistingof amino acid numbers 21 to 133 of the amino acid sequence of SEQ ID NO:36, or an antibody consisting of a heavy chain comprising a heavy chainvariable region sequence consisting of amino acid numbers 20 to 142 ofthe amino acid sequence of SEQ ID NO: 105 and a light chain comprising alight chain variable region sequence consisting of amino acid numbers 21to 133 of the amino acid sequence of SEQ ID NO: 36.

(35) The antibody according to (33), wherein the antibody is an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 472 of the amino acid sequence of SEQ ID NO:30 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 238 of the amino acid sequence of SEQ ID NO:36, or an antibody consisting of a heavy chain comprising an amino acidsequence consisting of amino acid numbers 20 to 468 of the amino acidsequence of SEQ ID NO: 105 and a light chain comprising an amino acidsequence consisting of amino acid numbers 21 to 238 of the amino acidsequence of SEQ ID NO: 36.

(36) The antibody according to (33), wherein the antibody is an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 468 of the amino acid sequence of SEQ ID NO:105 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 238 of the amino acid sequence of SEQ ID NO:36.

(37) An antibody or an antigen-binding fragment thereof whichspecifically binds to an extracellular region of ALK2 protein andinhibits ALK2-mediated BMP signal transduction, wherein the antibodycomprises

(a) a heavy chain variable region sequence selected from the groupconsisting of the following amino acid sequences:a1) an amino acid sequence consisting of amino acid numbers 20 to 140 ofthe amino acid sequence of SEQ ID NO: 40;a2) an amino acid sequence consisting of amino acid numbers 20 to 140 ofthe amino acid sequence of SEQ ID NO: 42;a3) an amino acid sequence consisting of amino acid numbers 20 to 140 ofthe amino acid sequence of SEQ ID NO: 44;a4) an amino acid sequence consisting of the amino acid numbers 20 to140 of the amino acid sequence of SEQ ID NO: 46;a5) an amino acid sequence consisting of amino acid numbers 20 to 140 ofan amino acid sequence of SEQ ID NO: 48;a6) an amino acid sequence consisting of amino acid numbers 20 to 140 ofthe amino acid sequence of SEQ ID NO: 107;a7) an amino acid sequence consisting of amino acid numbers 20 to 140 ofthe amino acid sequence of SEQ ID NO: 109;a8) an amino acid sequence having at least 95% identity to any one aminoacid sequence selected from the amino acid sequences a1) to a7);a9) an amino acid sequence having at least 99% identity to any one aminoacid sequence selected from the amino acid sequences a1) to a7); anda10) an amino acid sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several amino acid residues in any one aminoacid sequence selected from the amino acid sequences a1) to a7); and(b) a light chain variable region sequence selected from the groupconsisting of the following amino acid sequences:b1) an amino acid sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 50;b2) an amino acid sequence consisting of amino acid numbers 21 to 129 ofan amino acid sequence of SEQ ID NO: 52;b3) an amino acid sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 54;b4) an amino acid sequence consisting of amino acid numbers 21 to 129 ofan amino acid sequence of SEQ ID NO: 56;b5) an amino acid sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 58;b6) an amino acid sequence having at least 95% identity to any one aminoacid sequence selected from the amino acid sequences b1) to b5);b7) an amino acid sequence having at least 99% identity to any one aminoacid sequence selected from the amino acid sequences b1) to b5); andb8) an amino acid sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several amino acid residues in any one aminoacid sequence selected from the amino acid sequences b1) to b5).

(38) The antibody or the antigen-binding fragment thereof according to(37), wherein the antibody is an antibody consisting of a heavy chaincomprising a heavy chain variable region sequence consisting of aminoacid numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 42 and alight chain comprising a light chain variable region sequence consistingof amino acid numbers 21 to 129 of the amino acid sequence of SEQ ID NO:52, an antibody consisting of a heavy chain comprising a heavy chainvariable region sequence consisting of amino acid numbers 20 to 140 ofthe amino acid sequence of SEQ ID NO: 44 and a light chain comprising alight chain variable region sequence consisting of amino acid numbers 21to 129 of the amino acid sequence of SEQ ID NO: 56, an antibodyconsisting of a heavy chain comprising a heavy chain variable regionsequence consisting of amino acid numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 107 and a light chain comprising a light chainvariable region sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 52, or an antibody consisting of aheavy chain comprising a heavy chain variable region sequence consistingof amino acid numbers 20 to 140 of the amino acid sequence of SEQ ID NO:109 and a light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 129 of the amino acid sequence ofSEQ ID NO: 56.

(39) The antibody according to (37), wherein the antibody is an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 470 of the amino acid sequence of SEQ ID NO:42 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 234 of the amino acid sequence of SEQ ID NO:52, an antibody consisting of a heavy chain comprising an amino acidsequence consisting of amino acid numbers 20 to 470 of the amino acidsequence of SEQ ID NO: 44 and a light chain comprising an amino acidsequence consisting of amino acid numbers 21 to 234 of the amino acidsequence of SEQ ID NO: 56, an antibody consisting of a heavy chaincomprising an amino acid sequence consisting of amino acid numbers 20 to470 of the amino acid sequence of SEQ ID NO: 107 and a light chaincomprising an amino acid sequence consisting of amino acid numbers 21 to234 of the amino acid sequence of SEQ ID NO: 52, or an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 470 of the amino acid sequence of SEQ ID NO:109 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 234 of the amino acid sequence of SEQ ID NO:56.

(40) The antibody according to (37), wherein the antibody is an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 470 of the amino acid sequence of SEQ ID NO:107 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 234 of the amino acid sequence of SEQ ID NO:52, or an antibody consisting of a heavy chain comprising an amino acidsequence consisting of amino acid numbers 20 to 470 of the amino acidsequence of SEQ ID NO: 109 and a light chain comprising an amino acidsequence consisting of amino acid numbers 21 to 234 of the amino acidsequence of SEQ ID NO: 56.

(41) An antibody or an antigen-binding fragment thereof whichspecifically binds to an extracellular region of ALK2 protein andinhibits ALK2-mediated BMP signal transduction, wherein the antibodycomprises

(a) a heavy chain variable region sequence selected from the groupconsisting of the following amino acid sequences:a1) an amino acid sequence consisting of amino acid numbers 20 to 137 ofthe amino acid sequence of SEQ ID NO: 111;a2) an amino acid sequence consisting of amino acid numbers 20 to 137 ofthe amino acid sequence of SEQ ID NO: 113;a3) an amino acid sequence having at least 95% identity to the aminoacid sequence a1) or a2);a4) an amino acid sequence having at least 99% identity to the aminoacid sequence a1) or a2); anda5) an amino acid sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several amino acid residues in the aminoacid sequence a1) or a2); and(b) a light chain variable region sequence selected from the groupconsisting of the following amino acid sequences:b1) an amino acid sequence consisting of amino acid numbers 21 to 128 ofthe amino acid sequence of SEQ ID NO: 115;b2) an amino acid sequence consisting of amino acid numbers 21 to 128 ofthe amino acid sequence of SEQ ID NO: 117;b3) an amino acid sequence consisting of amino acid numbers 21 to 128 ofthe amino acid sequence of SEQ ID NO: 119;b4) an amino acid sequence having at least 95% identity to any one aminoacid sequence selected from the amino acid sequences b1) to b3);b5) an amino acid sequence having at least 99% identity to any one aminoacid sequence selected from the amino acid sequences b1) to b3); andb6) an amino acid sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several amino acid residues in any one aminoacid sequence selected from the amino acid sequences b1) to b3).

(42) An antibody or an antigen-binding fragment thereof whichspecifically binds to an extracellular region of ALK2 protein andinhibits ALK2-mediated BMP signal transduction, wherein the antibodycomprises

(a) a heavy chain variable region sequence selected from the groupconsisting of the following amino acid sequences:a1) an amino acid sequence consisting of amino acid numbers 20 to 137 ofthe amino acid sequence of SEQ ID NO: 121;a2) an amino acid sequence consisting of amino acid numbers 20 to 137 ofthe amino acid sequence of SEQ ID NO: 123;a3) an amino acid sequence having at least 95% identity to the aminoacid sequence a1) or a2);a4) an amino acid sequence having at least 99% identity to the aminoacid sequence a1) or a2); anda5) an amino acid sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several amino acid residues in the aminoacid sequence a1) or a2); and(b) a light chain variable region sequence selected from the groupconsisting of the following amino acid sequences:b1) an amino acid sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 125;b2) an amino acid sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 127;b3) an amino acid sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 129;b4) an amino acid sequence having at least 95% identity to any one aminoacid sequence selected from the amino acid sequences b1) to b3);b5) an amino acid sequence having at least 99% identity to any one aminoacid sequence selected from the amino acid sequences b1) to b3); andb6) an amino acid sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several amino acid residues in any one aminoacid sequence selected from the amino acid sequences b1) to b3).

(43) The antibody according to any one of (1) to (42), wherein theantibody is an antibody comprising a heavy chain in which one to severalcarboxyl-terminal amino acids are deleted.

(44) The antibody or the antigen-binding fragment thereof according toany one of (1) to (43), wherein the antibody is an antibody having apyroglutamylated amino-terminal amino acid residue in a heavy or lightchain thereof.

(45) A pharmaceutical composition comprising at least one antibody orantigen-binding fragment according to any one of (1) to (44).

(46) The pharmaceutical composition according to (45), wherein thepharmaceutical composition is a therapeutic and/or prophylactic drug forectopic ossification.

(47) A pharmaceutical composition for the treatment and/or prevention ofectopic ossification, comprising at least any one antibody orantigen-binding fragment thereof according to any one of (1) to (44) andat least any one drug selected from the group consisting ofanti-inflammatory drugs, steroids, bisphosphonates, muscle relaxants,and retinoic acid receptor (RAR) γ agonists.

(48) The pharmaceutical composition according to (46) or (47), whereinthe ectopic ossification is fibrodysplasia ossificans progressiva (FOP),progressive osseous heteroplasia (POH), traumatic ectopic ossification,or ectopic ossification after implant arthroplasty.

(49) The pharmaceutical composition according to (46) or (47), whereinthe ectopic ossification is fibrodysplasia ossificans progressiva (FOP).

(50) The pharmaceutical composition according to (45), wherein thepharmaceutical composition is a therapeutic and/or prophylactic drug foranemia.

(51) The pharmaceutical composition according to (45), wherein thepharmaceutical composition is a therapeutic and/or prophylactic drug fordiffuse intrinsic pontine glioma (DIPG).

(52) A method for treating and/or preventing ectopic ossification,comprising administering at least one antibody or antigen-bindingfragment according to any one of (1) to (44), or at least onepharmaceutical composition according to any one of (45) to (49).

(53) A method for treating and/or preventing ectopic ossification,comprising administering at least one antibody or antigen-bindingfragment thereof according to any one of (1) to (44), or at least onepharmaceutical composition according to any one of (45) to (49), and atleast any one drug selected from the group consisting ofanti-inflammatory drugs steroids, bisphosphonates, muscle relaxants, andretinoic acid receptor (RAR) γ agonists, concurrently (or, at the sametime) or separately (or, one after another).

(54) The method according to (52) or (53), wherein the ectopicossification is fibrodysplasia ossificans progressiva (FOP), progressiveosseous heteroplasia (POH), traumatic ectopic ossification, or ectopicossification after implant arthroplasty.

(55) The method according to (52) or (53), wherein the ectopicossification is fibrodysplasia ossificans progressiva (FOP).

(56) A method for treating and/or preventing anemia, comprisingadministering at least one antibody or antigen-binding fragmentaccording to any one of (1) to (44), or at least one pharmaceuticalcomposition according to (45) or (50).

(57) A method for treating and/or preventing diffuse intrinsic pontineglioma (DIPG), comprising administering at least one antibody orantigen-binding fragment according to any one of (1) to (44), or atleast one pharmaceutical composition according to (45) or (51).

(58) A polynucleotide encoding an antibody according to any one of (1)to (44).

(59) A polynucleotide comprising

(a) a polynucleotide selected from the group consisting of the followingnucleotide sequences:a1) a nucleotide sequence consisting of nucleotide numbers 58 to 426 ofthe nucleotide sequence of SEQ ID NO: 27;a2) a nucleotide sequence consisting of nucleotide numbers 58 to 426 ofthe nucleotide sequence of SEQ ID NO: 29;a3) a nucleotide sequence consisting of nucleotide numbers 58 to 426 ofthe nucleotide sequence of SEQ ID NO: 104;a4) a nucleotide sequence having at least 95% identity to any of thenucleotide sequences a1) to a3);a5) a nucleotide sequence having at least 99% identity to any of thenucleotide sequences a1) to a3);a6) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any of the nucleotide sequences a1) to a3);anda7) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in the nucleotidesequence a1) or a2); and/or(b) a polynucleotide selected from the group consisting of the followingnucleotide sequences:b1) a nucleotide sequence consisting of nucleotide numbers 86 to 424 ofthe nucleotide sequence of SEQ ID NO: 31;b2) a nucleotide sequence consisting of nucleotide numbers 86 to 424 ofthe nucleotide sequence of SEQ ID NO: 33;b3) a nucleotide sequence consisting of nucleotide numbers 86 to 424 ofthe nucleotide sequence of SEQ ID NO: 35;b4) a nucleotide sequence consisting of nucleotide numbers 86 to 424 ofthe nucleotide sequence of SEQ ID NO: 37;b5) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b4);b6) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b4);b7) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences b1) to b4); andb8) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequencesb1) to b4).

(60) A polynucleotide comprising

(a) a polynucleotide selected from the group consisting of the followingnucleotide sequences:a1) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 39;a2) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 41;a3) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 43;a4) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 45;a5) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 47;a6) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 106;a7) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 108;a8) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences a1) to a7);a9) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences a1) to a7);a10) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences a1) to a7); anda11) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequencesa1) to a7); and/or(b) a polynucleotide selected from the group consisting of the followingnucleotide sequences:b1) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 49;b2) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 51;b3) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 53;b4) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 55;b5) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 57;b6) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b5);b7) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b5);b8) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences b1) to b5); andb9) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequences b1) to b5).

(61) A polynucleotide comprising

(a) a polynucleotide selected from the group consisting of the followingnucleotide sequences:a1) a nucleotide sequence consisting of nucleotide numbers 58 to 411 ofthe nucleotide sequence of SEQ ID NO: 110;a2) a nucleotide sequence consisting of nucleotide numbers 58 to 411 ofthe nucleotide sequence of SEQ ID NO: 112;a3) a nucleotide sequence having at least 95% identity to the nucleotidesequence a1) or a2);a4) a nucleotide sequence having at least 99% identity to the nucleotidesequence a1) or a2);a5) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to the nucleotide sequence a1) or a2); anda6) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in the nucleotidesequence a1) or a2); and/or(b) a polynucleotide selected from the group consisting of the followingnucleotide sequences:b1) a nucleotide sequence consisting of nucleotide numbers 61 to 384 ofthe nucleotide sequence of SEQ ID NO: 114;b2) a nucleotide sequence consisting of nucleotide numbers 61 to 384 ofthe nucleotide sequence of SEQ ID NO: 116;b3) a nucleotide sequence consisting of nucleotide numbers 61 to 384 ofthe nucleotide sequence of SEQ ID NO: 118;b4) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b3);b5) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b3);b6) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences b1) to b3); andb7) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequencesb1) to b3).

(62) A polynucleotide comprising

(a) a polynucleotide selected from the group consisting of the followingnucleotide sequences:a1) a nucleotide sequence consisting of nucleotide numbers 58 to 411 ofthe nucleotide sequence of SEQ ID NO: 120;a2) a nucleotide sequence consisting of nucleotide numbers 58 to 411 ofthe nucleotide sequence of SEQ ID NO: 122;a3) a nucleotide sequence having at least 95% identity to the nucleotidesequence a1) or a2);a4) a nucleotide sequence having at least 99% identity to the nucleotidesequence a1) or a2);a5) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to the nucleotide sequence a1) or a2); anda6) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in the nucleotidesequence a1) or a2); and/or(b) a polynucleotide selected from the group consisting of the followingnucleotide sequences:b1) a nucleotide sequence consisting of nucleotide numbers 61 to 387 ofthe nucleotide sequence of SEQ ID NO: 124;b2) a nucleotide sequence consisting of nucleotide numbers 61 to 387 ofthe nucleotide sequence of SEQ ID NO: 126;b3) a nucleotide sequence consisting of nucleotide numbers 61 to 387 ofthe nucleotide sequence of SEQ ID NO: 128;b4) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b3);b5) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b3);b6) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences b1) to b3); andb7) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequencesb1) to b3).

(63) A vector comprising any one polynucleotide according to any one of(58) to (62).

(64) A transformed host cell comprising any one polynucleotide accordingto any one of (58) to (62).

(65) A transformed host cell comprising a vector according to (63).

(66) A method for producing an antibody according to any one of (1) to(44), comprising the step of culturing a host cell according to (64) or(65) and purifying the antibody from the culture product.

According to the present invention, a therapeutic and/or prophylacticdrug for ectopic ossification and/or bone dysplasia can be obtained.

The present specification encompasses the contents disclosed in JapanesePatent Application No. 2015-017882 from which the present applicationclaims the priority.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 This figure is a graph showing the recognition of mouseALK2-His&Fc (“mALK2-Fc”) by monoclonal antibodies produced by hybridomasA2-11E, A2-15A, A2-25C, and A2-27D, and the binding of these monoclonalantibodies to human Fc. A medium was used as a control (“Controlmedium”).

FIG. 2A This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-11E, A2-15A, A2-25C, and A2-27D do notrecognize cells expressing a fluorescent protein EGFP.

FIG. 2B This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-11E, A2-15A, A2-25C, and A2-27D recognizecells expressing wild-type mouse ALK2 (mALK2(WT)-EGFP).

FIG. 2C This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-11E, A2-15A, A2-25C, and A2-27D recognizecells expressing wild-type human ALK2 (hALK2(WT)-EGFP).

FIG. 2D This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-11E, A2-15A, A2-25C, and A2-27D recognizecells expressing human ALK2 having a FOP mutation (R206H)(hALK2(R206H)-EGFP).

FIG. 3 This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-11E, A2-15A, A2-25C, and A2-27Dspecifically recognize only ALK2-expressing cells and do not recognizecells expressing ALK, ALK3, or ALK6.

FIG. 4 This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-11E, A2-15A, A2-25C, and A2-27D recognizecells expressing wild-type ALK2 and cells expressing the shown 12 typesof ALK2 mutants identified in FOP.

FIG. 5 This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-11E, A2-15A, A2-25C, and A2-27D inhibit,in a dose-dependent manner, BMP-specific luciferase (luc) activityinduced by BMP7 in HEK293A cells in which wild-type ALK2 or a R206Hmutant has been overexpressed.

FIG. 6 This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-11E, A2-15A, A2-25C, and A2-27D cannotcompletely inhibit the BMP2-induced differentiation of C2C12 cells intoosteoblast-like cells.

FIG. 7 This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-11E, A2-15A, A2-25C, and A2-27D inhibit,in a dose-dependent manner, the BMP7- or GDF2/BMP9-induceddifferentiation of C2C12 cells into osteoblast-like cells.

FIG. 8 This figure is a graph showing that the monoclonal antibodiesproduced by the hybridomas A2-15A and A2-27D inhibit BMP7- orGDF2/BMP9-induced ectopic osteoinduction in mouse skeletal muscletissues.

FIG. 9 This figure is a graph showing that chimeric antibodies cA2-15Aand cA2-27D exhibit inhibitory activity equivalent to the rat monoclonalantibodies A2-15A and A2-27D, respectively, against BMP-specificluciferase (luc) activity induced by BMP7.

FIG. 10 This figure is a graph showing that the chimeric antibodiescA2-15A and cA2-27D inhibit, in a dose-dependent manner, the BMP-induceddifferentiation of C2C12 cells into osteoblast-like cells.

FIG. 11 This figure shows the amino acid sequence of each CDR sequenceof the A2-15A antibody.

FIG. 12 This figure shows the amino acid sequence of each CDR sequenceof the A2-27D antibody.

FIG. 13 This figure shows the amino acid sequence of each CDR sequenceof the A2-11E antibody.

FIG. 14 This figure shows the amino acid sequence of each CDR sequenceof the A2-25C antibody.

FIG. 15 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-15A-H1.

FIG. 16 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-15A-H4.

FIG. 17 This figure shows the nucleotide sequence of a DNA fragmentcomprising sequences encoding humanized hA2-15A-L1, and the amino acidsequence thereof.

FIG. 18 This figure shows the nucleotide sequence of a DNA fragmentcomprising sequences encoding humanized hA2-15A-L4, and the amino acidsequence thereof.

FIG. 19 This figure shows the nucleotide sequence of a DNA fragmentcomprising sequences encoding humanized hA2-15A-L6, and the amino acidsequence thereof.

FIG. 20 This figure shows the nucleotide sequence of a DNA fragmentcomprising sequences encoding humanized hA2-15A-L7, and the amino acidsequence thereof.

FIG. 21 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-27D-H1.

FIG. 22 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-27D-H2.

FIG. 23 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-27D-H3.

FIG. 24 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-27D-H4.

FIG. 25 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-27D-H5.

FIG. 26 This figure shows the nucleotide sequence of a DNA fragmentcomprising sequences encoding humanized hA2-27D-L1, and the amino acidsequence thereof.

FIG. 27 This figure shows the nucleotide sequence of a DNA fragmentcomprising sequences encoding humanized hA2-27D-L2, and the amino acidsequence thereof.

FIG. 28 This figure shows the nucleotide sequence of a DNA fragmentcomprising sequences encoding humanized hA2-27D-L3, and the amino acidsequence thereof.

FIG. 29 This figure shows the nucleotide sequence of a DNA fragmentcomprising sequences encoding humanized hA2-27D-L4, and the amino acidsequence thereof.

FIG. 30 This figure shows the nucleotide sequence of a DNA fragmentcomprising sequences encoding humanized hA2-27D-L5, and the amino acidsequence thereof.

FIG. 31 This figure is a graph showing that a humanized A2-15A antibody(IgG1) and humanized A2-27D antibodies (IgG1) inhibit, in adose-dependent manner, BMP-specific luciferase (luc) activity induced byBMP7.

FIG. 32 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-15A-H4 IgG2 type.

FIG. 33 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-27D-H2-LALA.

FIG. 34 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-27D-H3-LALA.

FIG. 35 This figure is a graph showing that a humanized A2-15A antibody(IgG2) and a humanized A2-27D antibody (LALA) inhibit, in adose-dependent manner, BMP-specific luciferase (luc) activity induced byBMP7.

FIG. 36 This figure is a graph showing that the humanized A2-15Aantibody (IgG2) and the humanized A2-27D antibody (LALA) inhibitBMP7-induced ectopic osteoinduction in mouse skeletal muscle tissues.

FIG. 37 This figure shows the X-ray crystal structure of a complex ofhuman ALK2-ECD and human chimeric cA2-27D-Fab.

FIG. 38 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-11E-H3.

FIG. 39 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-11E-H4.

FIG. 40 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-11E-L2.

FIG. 41 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-11E-L3.

FIG. 42 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-11E-L4.

FIG. 43 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-25C-H3.

FIG. 44 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-25C-H4.

FIG. 45 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-25C-L 1.

FIG. 46 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-25C-L2.

FIG. 47 This figure shows the nucleotide sequence and amino acidsequence of humanized hA2-25C-L3.

FIG. 48 This figure is a graph showing that humanized A2-11E antibodies(IgG1) and humanized A2-25C antibodies (IgG1) inhibit, in adose-dependent manner, the BMP-induced differentiation of C2C12 cellsinto osteoblast-like cells.

FIG. 49 This figure shows the X-ray crystal structure of a complex ofhuman ALK2-ECD and human chimeric cA2-25C-Fab.

FIG. 50 This figure is a graph showing that the monoclonal antibodyproduced by the hybridoma A2-27D inhibits BMP7-induced luciferase (Luc)activity in wild-type ALK2 (WT) and all of the shown 13 types ofmutants.

MODES FOR CARRYING OUT THE INVENTION 1. Definition

As used herein, the term “gene” includes not only DNA but mRNA, cDNA,and cRNA.

As used herein, the term “polynucleotide” is used with the same meaningas a nucleic acid and also includes DNA, RNA, probes, oligonucleotides,and primers.

As used herein, the “polypeptide” and the “protein” are usedinterchangeably with each other.

As used herein, the “RNA fraction” refers to a fraction containing RNA.

As used herein, the “cell” also includes cells within animal individualsand cultured cells.

As used herein, “ALK2” is used with the same meaning as ALK2 protein andincludes wild-type ALK2 and mutants (also referred to as mutant ALK2).

As used herein, the “antigen-binding fragment of an (the) antibody”,also called “functional fragment of an (the) antibody”, means a partialfragment of the antibody having an activity binding to the antigen andincludes, for example, Fab, F(ab′)2, Fv, scFv, diabodies, linearantibodies, and multispecific antibodies formed from antibody fragments.The antigen-binding fragment of an antibody also includes Fab′, which isa monovalent fragment of antibody variable regions obtained by treatmentof F(ab′)2 under reducing conditions. However, the antigen-bindingfragment of an antibody is not limited to these molecules as long as theantigen-binding fragment has the ability to bind to the antigen. Such anantigen-binding fragment includes not only a fragment obtained bytreating a full-length molecule of the antibody protein with anappropriate enzyme but a protein produced in appropriate host cellsusing a genetically engineered antibody gene.

As used herein, the “epitope”, also called “antigenic determinant”,generally refers to an antibody-binding antigenic site consisting of atleast 7 amino acids, at least 8 amino acids, at least 9 amino acids, orat least 10 amino acids, of an antigen. As used herein, the epitopemeans a partial peptide or a partial conformation of ALK2 to which aparticular anti-ALK2 antibody binds. The epitope as a partial peptide ofALK2 can be determined by a method well known to those skilled in theart such as immunoassay and can be determined, for example, by thefollowing method: various partial structures of ALK2 are prepared. Forthe preparation of the partial structures, an oligopeptide synthesistechnique known in the art can be used. For example, a series ofpolypeptide fragments having an appropriate length are prepared in orderfrom the C or N terminus of ALK2 by use of a gene recombinationtechnique well known to those skilled in the art. Then, the reactivityof the antibody therewith is studied to roughly determine a recognitionsite. Then, shorter peptides are synthesized, and the reactivity of theantibody with these peptides can be studied to determine the epitope.Alternatively, the epitope as a partial conformation of ALK2 to which aparticular ALK2 antibody binds can be determined by identifying aminoacid residues of ALK2 adjacent to the antibody by X-ray crystalstructure analysis. Provided that a second anti-ALK2 antibody binds to apartial peptide or a partial conformation that is bound by a firstanti-ALK2 antibody, the first antibody and the second antibody can bedetermined to share an epitope. If the specific sequence or structure ofan epitope is not determined, the first antibody and the second antibodycan be determined to share the epitope by confirming that the secondanti-ALK2 antibody (cross-)competes with the first anti-ALK2 antibodyfor binding to ALK2 (i.e., that the second antibody interferes with thebinding of the first antibody to ALK2). When the first antibody and thesecond antibody bind to a common epitope and the first antibody has anactivity such as inhibitory activity against ALK2-mediated BMP signals,the second antibody can also be expected to have similar activity.

The heavy and light chains of an antibody molecule are known to eachhave three complementarity determining regions (CDRs). Thecomplementarity determining regions, also called hypervariable domains,are located in the variable regions of the antibody heavy and lightchains. These sites have a particularly highly variable primarystructure and are separated at three places on the respective primarystructures of heavy and light chain polypeptide chains. As used herein,the complementarity determining regions of an antibody are referred toas CDRH1, CDRH2, and CDRH3 from the amino terminus of the heavy chainamino acid sequence for the complementarity determining regions of theheavy chain and as CDRL1, CDRL2, and CDRL3 from the amino terminus ofthe light chain amino acid sequence for the complementarity determiningregions of the light chain. These sites are proximal to each other onthe conformation and determine specificity for the antigen to be bound.

In the present invention, the wording “hybridizing under stringentconditions” means hybridization under conditions involving hybridizationat approximately 50 to 70° C. (e.g., 68° C.) in a commercially availablehybridization solution ExpressHyb Hybridization Solution (manufacturedby Clontech Laboratories, Inc.), or hybridization at approximately 50 to70° C. (e.g., 68° C.) in the presence of approximately 0.7 to 1.0 M NaClusing a DNA-immobilized filter, followed by washing at approximately 50to 70° C. (e.g., 68° C.) using an SSC solution having an approximately0.1 to 2× concentration (SSC having a 1× concentration consists of 150mM NaCl and 15 mM sodium citrate; if necessary, the solution may containapproximately 0.1 to 0.5% SDS) which permits identification, orhybridization under conditions equivalent thereto.

As used herein, the term “several” in the phrase “one to several” and“one or several” refers to 2 to 10. The term “several” is preferably 10or less, more preferably 5 or 6 or less, further preferably 2 or 3.

2. ALK2

The ALK2 gene is a responsible gene for FOP encoding a receptor of BMPthat induces ectopic bone formation in soft tissues including skeletalmuscle tissues. Mutant ALK2 having amino acid substitutions has beenfound from familial and sporadic FOP cases. For example, L196P (mutationthat substitutes leucine at position 196 by proline), delP197_F198insL(mutation that deletes proline at position 197 and phenylalanine atposition 198 and inserts leucine), R202I (mutation that substitutesarginine at position 202 by isoleucine), R206H (mutation thatsubstitutes arginine at position 206 by histidine), Q207E (mutation thatsubstitutes glutamine at position 207 by glutamic acid), R258S (mutationthat substitutes arginine at position 258 by serine), R258G (mutationthat substitutes arginine at position 258 by glycine), G325A (mutationthat substitutes glycine at position 325 by alanine), G328E (mutationthat substitutes glycine at position 328 by glutamic acid), G328R(mutation that substitutes glycine at position 328 by arginine), G328W(mutation that substitutes glycine at position 328 by tryptophan), G356D(mutation that substitutes glycine at position 356 by aspartic acid),and R375P (mutation that substitutes arginine at position 375 byproline) mutants are known as mutants of human ALK2.

ALK2 used in the present invention can be obtained by in vitro synthesisor by production from host cells through gene manipulation.Specifically, ALK2 cDNA is inserted into a vector that permitsexpression. Then, the ALK2 protein can be obtained by synthesis in asolution containing enzymes, substrates, and energy substances necessaryfor transcription and translation, or by expression from otherprokaryotic or eukaryotic host cells transformed with the vector.

ALK2 used in the present invention is ALK2 derived from a mammalincluding a human or a mouse. For example, the amino acid sequence andnucleotide sequence of human ALK2 are available with reference toGenBank Accession No. NM-001105. Herein, the amino acid sequence is alsodisclosed as SEQ ID NO: 84, and the nucleotide sequence is disclosed asSEQ ID NO: 85. The amino acid sequence and nucleotide sequence of mouseALK2 are available with reference to GenBank Accession No. NP-001103674.Herein the amino acid sequence is also disclosed as SEQ ID NO: 86, andthe nucleotide sequence is disclosed as SEQ ID NO: 87. ALK2 is alsocalled ACVR1 (activin A type I receptor 1) or ACTR1 (activin receptortype 1), and all of these terms represent the same molecules.

The ALK2 cDNA can be obtained by a so-called PCR method which involvescarrying out polymerase chain reaction (hereinafter, referred to as“PCR”) (Saiki, R. K., et al., Science, (1988) 239, 487-49), for example,with a cDNA library expressing the ALK2 cDNA as a template using primersspecifically amplifying the ALK2 cDNA.

The ALK2 cDNA also includes a polynucleotide that hybridizes understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to the nucleotide sequence encoding human ormouse ALK2 and encodes a protein having biological activity equivalentto ALK2. The ALK2 cDNA further includes a splicing variant that has beentranscribed from the human or mouse ALK2 gene locus or a polynucleotidethat hybridizes under stringent conditions thereto, and encodes aprotein having biological activity equivalent to ALK2.

ALK2 also includes a protein that consists of an amino acid sequencederived from the amino acid sequence of human or mouse ALK2 or an aminoacid sequence thereof free from the signal sequence by the substation,deletion, or addition of one or several amino acids, and has biologicalactivity equivalent to ALK2. ALK2 further includes a protein thatconsists of an amino acid sequence encoded by a splicing varianttranscribed from the human or mouse ALK2 gene locus, or an amino acidsequence derived from this amino acid sequence by the substation,deletion, or addition of one or several amino acids, and has biologicalactivity equivalent to ALK2.

3. Detection of Ectopic Ossification and/or Bone Dysplasia

Ectopic ossification and/or bone dysplasia is induced by ALK2-mediatedBMP signal transduction.

The “ectopic ossification” means bone formation at a site where the boneis originally absent. The “bone dysplasia” means the abnormal shape orquality of the existing bone. Examples of the “ectopic ossification” caninclude, but are not limited to, fibrodysplasia ossificans progressiva(FOP), progressive osseous heteroplasia (POH), traumatic ectopicossification, and ectopic ossification after implant arthroplasty.Examples of the “bone dysplasia” can include, but are not limited to,spondyloarthritis (SpA) and ankylosing spondylitis (AS).

ALK2 is a transmembrane serine-threonine kinase receptor binding to BMP.ALK2 binds to BMP at the N-terminal extracellular region and activates adownstream intracellular signal transduction system throughintracellular serine-threonine kinase. Bone morphogenetic protein (BMP)is a multifunctional growth factor belonging to the transforming growthfactor β (TGF-β) superfamily, and approximately 20 BMP family membershave been identified. BMP has been confirmed to induce ectopic boneformation in soft tissues including skeletal muscle tissues and istherefore considered to participate in diseases promoting abnormal boneformation. BMP-2 and BMP-4 are considered to have higher affinity forALK3 than that for ALK2. Since ALK3 is expressed ubiquitously ascompared with ALK2, BMP-2 or BMP-4 seems to be often used in general inexperiments of inducing ectopic ossification at various sites. On theother hand, BMP-7 has relatively high affinity for ALK2. BMP-9 isgenerally considered to have high affinity for ALK1 and has also beenfound to have relatively high affinity for ALK2. In FOP, ectopicossification occurs via ALK2. Therefore, the presence or absence oftherapeutic and/or prophylactic effects on FOP can probably be confirmedby testing efficacy for ectopic osteoinduction caused by the activationof ALK2-mediated signals by BMP-7 and BMP-9.

The culture of myoblasts (C2C12 cells) in the presence of BMP suppressestheir differentiation into mature muscle cells through an intracellularsignal transduction mechanism specific for BMP and instead induces thedifferentiation into osteoblasts. Thus, ALK2-mediated BMP signaltransduction can be analyzed with models of induction of differentiationof C2C12 cells into osteoblasts by BMP.

4. Production of Anti-ALK2 Antibody

The antibody of the present invention against ALK2 can be obtained by amethod described in, for example, WO2009/091048, WO2011/027808, orWO2012/133572. Specifically, nonhuman animals are immunized with theantigen of interest. Lymphs, lymph tissues, blood samples, or bonemarrow-derived cells are collected from the animals after establishmentof immunity. Plasma cells and/or plasmablasts of the nonhuman animalsspecifically binding to the antigen of interest are selected. A gene ofan antibody to the antigen of interest is collected from the obtainedplasma cells and/or plasmablasts, and its nucleotide sequence isidentified. The antibody or a fragment thereof can be obtained on thebasis of the identified nucleotide sequence of the gene. Alternatively,the antibody or the antibody fragment can be obtained by obtainingplasma cells and/or plasmablasts in the same way as above from the bloodof a human infected patient. The obtained antibody can be tested for itsbinding activity to ALK2 to select an antibody applicable to humandiseases. Examples of the monoclonal antibody thus obtained can includeA2-11E, A2-15A, A2-25C, and A2-27D.

Herein, amino acid numbers assigned to CDR/FR characteristic of anantibody are laid out according to the KABAT numbering (KABAT et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService National Institutes of Health, Bethesda, Md. (1991)).

The monoclonal antibody can also be obtained according to a method knownin the art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497;and Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press,N.Y. (1980)) by fusing antibody-producing cells that produce theantibody against ALK2 with myeloma cells to establish hybridomas.Specific examples of such a method are described in WO2009/48072(published on Apr. 16, 2009) and WO2010/117011 (published on Oct. 14,2010). However, the method for obtaining monoclonal antibodiescorresponds to an already established field and is not limited to thespecific examples described above.

The antibody of the present invention includes monoclonal antibodiesagainst ALK2 described above as well as, for example, polyclonalantibodies similarly having therapeutic and/or prophylactic effects,recombinant antibodies artificially engineered for the purpose of, forexample, reducing heterogeneous antigenicity against humans, forexample, chimeric antibodies, humanized antibodies, human antibodies,and the like. These antibodies can be produced by use of known methods.

Examples of the chimeric antibody can include chimeric antibodiescomprising variable regions and constant regions (Fc) of antibodiesderived from different species, for example, the variable regions of amouse- or rat-derived antibody joined to human-derived constant regions(see Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984)). One exampleof the chimeric antibody derived from A2-15A can include an antibodyconsisting of a heavy chain having an amino acid sequence consisting ofamino acid numbers 20 to 472 of SEQ ID NO: 20 of the Sequence Listingand a light chain having an amino acid sequence consisting of aminoacids 21 to 237 of SEQ ID NO: 22. One example of the chimeric antibodyderived from A2-27D can include an antibody consisting of a heavy chainhaving an amino acid sequence consisting of amino acids 20 to 470 of SEQID NO: 24 of the Sequence Listing and a light chain having an amino acidsequence consisting of amino acids 21 to 234 of SEQ ID NO: 26.

Examples of the humanized antibody can include an antibody comprisingCDRs alone integrated into a human-derived antibody (see Nature (1986)321, p. 522-525), and an antibody comprising the CDR sequences as wellas amino acid residues of a portion of frameworks grafted into a humanantibody by a CDR grafting method (International Publication No.WO90/07861).

The humanized antibody derived from the A2-15A antibody is included inthe antibody of the present invention as long as the humanized antibodycontains all of the 6 CDR sequences of A2-15A and has binding activityagainst ALK2. The heavy chain variable region of the A2-15A antibodycomprises CDRH1 consisting of the amino acid sequence of SEQ ID NO: 59(GFTFSHYYMA), CDRH2 consisting of the amino acid sequence of SEQ ID NO:60 (SITNSGGSINYRDSVKG), and CDRH3 consisting of the amino acid sequenceof SEQ ID NO: 61 (EGGENYGGYPPFAY). The light chain variable region ofthe A2-15A antibody comprises CDRL1 consisting of the amino acidsequence of SEQ ID NO: 62 (RANQGVSLSRYNLMH), CDRL2 consisting of theamino acid sequence of SEQ ID NO: 63 (RSSNLAS), and CDRL3 consisting ofthe amino acid sequence of SEQ ID NO: 64 (QQSRESPFT). The amino acidsequences of these CDRs are also described in FIG. 11.

The humanized antibody derived from the A2-27D antibody is included inthe antibody of the present invention as long as the humanized antibodycontains all of the 6 CDR sequences of A2-27D and has binding activityagainst ALK2. The heavy chain variable region of the A2-27D antibodycomprises CDRH1 consisting of the amino acid sequence of SEQ ID NO: 65(GSTFSNYGMK), CDRH2 consisting of the amino acid sequence of SEQ ID NO:66 (SISRSSTYIYYADTVKG), and CDRH3 consisting of the amino acid sequenceof SEQ ID NO: 67 (AISTPFYWYFDF). The light chain variable region of theA2-27D antibody comprises CDRL1 consisting of the amino acid sequence ofSEQ ID NO: 68 (LASSSVSYMT), CDRL2 consisting of the amino acid sequenceof SEQ ID NO: 69 (GTSNLAS), and CDRL3 consisting of the amino acidsequence of SEQ ID NO: 70 (LHLTSYPPYT). The amino acid sequences ofthese CDRs are also described in FIG. 12.

The humanized antibody derived from the A2-11E antibody is included inthe antibody of the present invention as long as the humanized antibodycontains all of the 6 CDR sequences of A2-11E and has binding activityagainst ALK2. The heavy chain variable region of the A2-11E antibodycomprises CDRH1 consisting of the amino acid sequence of SEQ ID NO: 72(GFTFSNYYMY), CDRH2 consisting of the amino acid sequence of SEQ ID NO:73 (SINTDGGSTYYPDSVKG), and CDRH3 consisting of the amino acid sequenceof SEQ ID NO: 74 (STPNIPLAY). The light chain variable region of theA2-11E antibody comprises CDRL1 consisting of the amino acid sequence ofSEQ ID NO: 75 (KASQNIYKYLN), CDRL2 consisting of the amino acid sequenceof SEQ ID NO: 76 (YSNSLQT), and CDRL3 consisting of the amino acidsequence of SEQ ID NO: 77 (FQYSSGPT). The amino acid sequences of theseCDRs are also described in FIG. 13.

The humanized antibody derived from the A2-25C antibody is included inthe antibody of the present invention as long as the humanized antibodycontains all of the 6 CDR sequences of A2-25C and has binding activityagainst ALK2. The heavy chain variable region of the A2-25C antibodycomprises CDRH1 consisting of the amino acid sequence of SEQ ID NO: 78(GFTFSYYAMS), CDRH2 consisting of the amino acid sequence of SEQ ID NO:79 (SISRGGDNTYYRDTVKG), and CDRH3 consisting of the amino acid sequenceof SEQ ID NO: 80 (LNYNNYFDY). The light chain variable region of theA2-25C antibody comprises CDRL1 consisting of the amino acid sequence ofSEQ ID NO: 81 (QASQDIGNWLS), CDRL2 consisting of the amino acid sequenceof SEQ ID NO: 82 (GATSLAD), and CDRL3 consisting of the amino acidsequence of SEQ ID NO: 83 (LQAYSAPFT). The amino acid sequences of theseCDRs are also described in FIG. 14.

A CDR-engineered humanized antibody prepared by substitution of 1 to 3amino acid residues in each CDR by other amino acid residues is alsoincluded in the antibody of the present invention as long as thehumanized antibody has binding activity against ALK2. Examples of theamino acid substitution in CDRL2 can include the substitution of oneamino acid of CDRL2 in the amino acid sequence of SEQ ID NO: 34. CDRL2consisting of the amino acid sequence of SEQ ID NO: 71 (RSSNLAQ) ispreferred. The amino acid sequence of this CDR is also described in FIG.11.

Actual examples of the humanized antibody derived from the A2-15Aantibody can include an arbitrary combination of a heavy chaincomprising a heavy chain variable region consisting of an amino acidsequence consisting of amino acid numbers 20 to 142 of the amino acidsequence of SEQ ID NO: 28 of the Sequence Listing, an amino acidsequence consisting of amino acid numbers 20 to 142 of the amino acidsequence of SEQ ID NO: 30, or an amino acid sequence consisting of aminoacid numbers 20 to 142 of the amino acid sequence of SEQ ID NO: 105 ofthe Sequence Listing, and a light chain comprising a light chainvariable region consisting of an amino acid sequence consisting of aminoacid numbers 21 to 133 of the amino acid sequence of SEQ ID NO: 32, anamino acid sequence consisting of amino acid numbers 21 to 133 of theamino acid sequence of SEQ ID NO: 34, an amino acid sequence consistingof amino acid numbers 21 to 133 of the amino acid sequence of SEQ ID NO:36, or an amino acid sequence consisting of amino acid numbers 21 to 133of the amino acid sequence of SEQ ID NO: 38.

Preferred examples of the combination can include an antibody consistingof a heavy chain comprising a heavy chain variable region sequenceconsisting of amino acid numbers 20 to 142 of the amino acid sequence ofSEQ ID NO: 28 and a light chain comprising a light chain variable regionsequence consisting of amino acid numbers 21 to 133 of the amino acidsequence of SEQ ID NO: 32, an antibody consisting of a heavy chaincomprising a heavy chain variable region sequence consisting of aminoacid numbers 20 to 142 of the amino acid sequence of SEQ ID NO: 28 and alight chain comprising a light chain variable region sequence consistingof amino acid numbers 21 to 133 of the amino acid sequence of SEQ ID NO:34, an antibody consisting of a heavy chain comprising a heavy chainvariable region sequence consisting of amino acid numbers 20 to 142 ofan amino acid sequence of SEQ ID NO: 30 and a light chain comprising alight chain variable region sequence consisting of amino acid numbers 21to 133 of the amino acid sequence of SEQ ID NO: 32, an antibodyconsisting of a heavy chain comprising a heavy chain variable regionsequence consisting of amino acid numbers 20 to 142 of the amino acidsequence of SEQ ID NO: 30 and a light chain comprising a light chainvariable region sequence consisting of amino acid numbers 21 to 133 ofthe amino acid sequence of SEQ ID NO: 34, an antibody consisting of aheavy chain comprising a heavy chain variable region sequence consistingof amino acid numbers 20 to 142 of the amino acid sequence of SEQ ID NO:30 and a light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 133 of the amino acid sequence ofSEQ ID NO: 36, an antibody consisting of a heavy chain comprising aheavy chain variable region sequence consisting of amino acid numbers 20to 142 of the amino acid sequence of SEQ ID NO: 30 and a light chaincomprising a light chain variable region sequence consisting of aminoacid numbers 21 to 133 of the amino acid sequence of SEQ ID NO: 38, andan antibody consisting of a heavy chain comprising a heavy chainvariable region sequence consisting of amino acid numbers 20 to 142 ofthe amino acid sequence of SEQ ID NO: 105 and a light chain comprising alight chain variable region sequence consisting of amino acid numbers 21to 133 of the amino acid sequence of SEQ ID NO: 36.

More preferred examples of the combination can include an antibodyconsisting of a heavy chain having an amino acid sequence consisting ofamino acid numbers 20 to 472 of the amino acid sequence of SEQ ID NO: 28and a light chain comprising an amino acid sequence consisting of aminoacid numbers 21 to 238 of the amino acid sequence of SEQ ID NO: 32, anantibody consisting of a heavy chain comprising an amino acid sequenceconsisting of amino acid numbers 20 to 472 of the amino acid sequence ofSEQ ID NO: 28 and a light chain comprising an amino acid sequenceconsisting of amino acid numbers 21 to 238 of the amino acid sequence ofSEQ ID NO: 34, an antibody consisting of a heavy chain comprising anamino acid sequence consisting of amino acid numbers 20 to 472 of theamino acid sequence of SEQ ID NO: 30 and a light chain comprising anamino acid sequence consisting of amino acid numbers 21 to 238 of theamino acid sequence of SEQ ID NO: 32, an antibody consisting of a heavychain comprising an amino acid sequence consisting of amino acid numbers20 to 472 of the amino acid sequence of SEQ ID NO: 30 and a light chaincomprising an amino acid sequence consisting of amino acid numbers 21 to238 of the amino acid sequence of SEQ ID NO: 34, an antibody consistingof a heavy chain comprising an amino acid sequence consisting of aminoacid numbers 20 to 472 of the amino acid sequence of SEQ ID NO: 30 and alight chain comprising an amino acid sequence consisting of amino acidnumbers 21 to 238 of the amino acid sequence of SEQ ID NO: 36, anantibody consisting of a heavy chain comprising an amino acid sequenceconsisting of amino acid numbers 20 to 472 of the amino acid sequence ofSEQ ID NO: 30 and a light chain comprising an amino acid sequenceconsisting of amino acid numbers 21 to 238 of the amino acid sequence ofSEQ ID NO: 38, and an antibody consisting of a heavy chain comprising aheavy chain variable region sequence consisting of amino acid numbers 20to 468 of the amino acid sequence of SEQ ID NO: 105 and a light chaincomprising a light chain variable region sequence consisting of aminoacid numbers 21 to 238 of the amino acid sequence of SEQ ID NO: 36.

Further preferred examples of the combination can include an antibodyconsisting of a heavy chain comprising a heavy chain variable regionsequence consisting of amino acid numbers 20 to 142 of the amino acidsequence of SEQ ID NO: 30 and a light chain comprising a light chainvariable region sequence consisting of amino acid numbers 21 to 133 ofthe amino acid sequence of SEQ ID NO: 36, and an antibody consisting ofa heavy chain comprising a heavy chain variable region sequenceconsisting of amino acid numbers 20 to 142 of the amino acid sequence ofSEQ ID NO: 105 and a light chain comprising a light chain variableregion sequence consisting of amino acid numbers 21 to 133 of the aminoacid sequence of SEQ ID NO: 36.

Still further preferred examples of the combination can include anantibody consisting of a heavy chain comprising an amino acid sequenceconsisting of amino acid numbers 20 to 472 of the amino acid sequence ofSEQ ID NO: 30 and a light chain comprising an amino acid sequenceconsisting of amino acid numbers 21 to 238 of the amino acid sequence ofSEQ ID NO: 36, and an antibody consisting of a heavy chain comprising anamino acid sequence consisting of amino acid numbers 20 to 468 of theamino acid sequence of SEQ ID NO: 105 and a light chain comprising anamino acid sequence consisting of amino acid numbers 21 to 238 of theamino acid sequence of SEQ ID NO: 36.

Most preferred examples of the combination can include an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 468 of the amino acid sequence of SEQ ID NO:105 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 238 of the amino acid sequence of SEQ ID NO:36.

Actual examples of the humanized antibody derived from the A2-27Dantibody can include an arbitrary combination of a heavy chaincomprising a heavy chain variable region consisting of an amino acidsequence consisting of amino acid numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 40 of the Sequence Listing, an amino acidsequence consisting of amino acid numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 42 of the Sequence Listing, an amino acidsequence consisting of amino acid numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 44 of the Sequence Listing, an amino acidsequence consisting of amino acid numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 46 of the Sequence Listing, an amino acidsequence consisting of amino acid numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 48, an amino acid sequence consisting of aminoacid numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 107, oran amino acid sequence consisting of amino acid numbers 20 to 140 of theamino acid sequence of SEQ ID NO: 109, and a light chain comprising alight chain variable region consisting of an amino acid sequenceconsisting of amino acid numbers 21 to 129 of the amino acid sequence ofSEQ ID NO: 50, an amino acid sequence consisting of amino acid numbers21 to 129 of the amino acid sequence of SEQ ID NO: 52, an amino acidsequence consisting of amino acid numbers 21 to 129 of the amino acidsequence of SEQ ID NO: 54, an amino acid sequence consisting of aminoacid numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 56, oran amino acid sequence consisting of amino acid numbers 21 to 129 of theamino acid sequence of SEQ ID NO: 58.

Preferred examples of the combination can include an antibody consistingof a heavy chain comprising a heavy chain variable region sequenceconsisting of amino acid numbers 20 to 140 of the amino acid sequence ofSEQ ID NO: 40 and a light chain comprising a light chain variable regionsequence consisting of amino acid numbers 21 to 129 of the amino acidsequence of SEQ ID NO: 50, an antibody consisting of a heavy chaincomprising a heavy chain variable region sequence consisting of aminoacid numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 40 and alight chain comprising a light chain variable region sequence consistingof amino acid numbers 21 to 129 of the amino acid sequence of SEQ ID NO:52, an antibody consisting of a heavy chain comprising a heavy chainvariable region sequence consisting of amino acid numbers 20 to 140 ofan amino acid sequence of SEQ ID NO: 40 and a light chain comprising alight chain variable region sequence consisting of amino acid numbers 21to 129 of the amino acid sequence of SEQ ID NO: 54, an antibodyconsisting of a heavy chain comprising a heavy chain variable regionsequence consisting of amino acid numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 42 and a light chain comprising a light chainvariable region sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 50, an antibody consisting of aheavy chain comprising a heavy chain variable region sequence consistingof amino acid numbers 20 to 140 of an amino acid sequence of SEQ ID NO:42 and a light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 129 of the amino acid sequence ofSEQ ID NO: 52, an antibody consisting of a heavy chain comprising aheavy chain variable region sequence consisting of amino acid numbers 20to 140 of an amino acid sequence of SEQ ID NO: 42 and a light chaincomprising a light chain variable region sequence consisting of aminoacid numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 54, anantibody consisting of a heavy chain comprising a heavy chain variableregion sequence consisting of amino acid numbers 20 to 140 of the aminoacid sequence of SEQ ID NO: 44 and a light chain comprising a lightchain variable region sequence consisting of amino acid numbers 21 to129 of the amino acid sequence of SEQ ID NO: 50, an antibody consistingof a heavy chain comprising a heavy chain variable region sequenceconsisting of amino acid numbers 20 to 140 of the amino acid sequence ofSEQ ID NO: 44 and a light chain comprising a light chain variable regionsequence consisting of amino acid numbers 21 to 129 of the amino acidsequence of SEQ ID NO: 52, an antibody consisting of a heavy chaincomprising a heavy chain variable region sequence consisting of aminoacid numbers 20 to 140 of an amino acid sequence of SEQ ID NO: 44 and alight chain comprising a light chain variable region sequence consistingof amino acid numbers 21 to 129 of the amino acid sequence of SEQ ID NO:54, an antibody consisting of a heavy chain comprising a heavy chainvariable region sequence consisting of amino acid numbers 20 to 140 ofthe amino acid sequence of SEQ ID NO: 44 and a light chain comprising alight chain variable region sequence consisting of amino acid numbers 21to 129 of the amino acid sequence of SEQ ID NO: 56, an antibodyconsisting of a heavy chain comprising a heavy chain variable regionsequence consisting of amino acid numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 46 and a light chain comprising a light chainvariable region sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 54, an antibody consisting of aheavy chain comprising a heavy chain variable region sequence consistingof amino acid numbers 20 to 140 of the amino acid sequence of SEQ ID NO:46 and a light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 129 of the amino acid sequence ofSEQ ID NO: 56, an antibody consisting of a heavy chain comprising aheavy chain variable region sequence consisting of amino acid numbers 20to 140 of the amino acid sequence of SEQ ID NO: 46 and a light chaincomprising a light chain variable region sequence consisting of aminoacid numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 58, anantibody consisting of a heavy chain comprising a heavy chain variableregion sequence consisting of amino acid numbers 20 to 140 of the aminoacid sequence of SEQ ID NO: 48 and a light chain comprising a lightchain variable region sequence consisting of amino acid numbers 21 to129 of the amino acid sequence of SEQ ID NO: 56, an antibody consistingof a heavy chain comprising a heavy chain variable region sequenceconsisting of amino acid numbers 20 to 140 of the amino acid sequence ofSEQ ID NO: 107 and a light chain comprising a light chain variableregion sequence consisting of amino acid numbers 21 to 129 of the aminoacid sequence of SEQ ID NO: 52, and an antibody consisting of a heavychain comprising a heavy chain variable region sequence consisting ofamino acid numbers 20 to 140 of the amino acid sequence of SEQ ID NO:109 and a light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 129 of the amino acid sequence ofSEQ ID NO: 56.

More preferred examples of the combination can include an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 470 of the amino acid sequence of SEQ ID NO:40 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 234 of the amino acid sequence of SEQ ID NO:50, an antibody consisting of a heavy chain comprising an amino acidsequence consisting of amino acid numbers 20 to 470 of the amino acidsequence of SEQ ID NO: 40 and a light chain comprising an amino acidsequence consisting of amino acid numbers 21 to 234 of the amino acidsequence of SEQ ID NO: 52, an antibody consisting of a heavy chaincomprising an amino acid sequence consisting of amino acid numbers 20 to470 of the amino acid sequence of SEQ ID NO: 40 and a light chaincomprising an amino acid sequence consisting of amino acid numbers 21 to234 of the amino acid sequence of SEQ ID NO: 54, an antibody consistingof a heavy chain comprising an amino acid sequence consisting of aminoacid numbers 20 to 470 of the amino acid sequence of SEQ ID NO: 42 and alight chain comprising an amino acid sequence consisting of amino acidnumbers 21 to 234 of the amino acid sequence of SEQ ID NO: 50, anantibody consisting of a heavy chain comprising an amino acid sequenceconsisting of amino acid numbers 20 to 470 of the amino acid sequence ofSEQ ID NO: 42 and a light chain comprising an amino acid sequenceconsisting of amino acid numbers 21 to 234 of the amino acid sequence ofSEQ ID NO: 52, an antibody consisting of a heavy chain comprising anamino acid sequence consisting of amino acid numbers 20 to 470 of theamino acid sequence of SEQ ID NO: 42 and a light chain comprising anamino acid sequence consisting of amino acid numbers 21 to 234 of theamino acid sequence of SEQ ID NO: 54, an antibody consisting of a heavychain comprising an amino acid sequence consisting of amino acid numbers20 to 470 of the amino acid sequence of SEQ ID NO: 44 and a light chaincomprising an amino acid sequence consisting of amino acid numbers 21 to234 of the amino acid sequence of SEQ ID NO: 50, an antibody consistingof a heavy chain comprising an amino acid sequence consisting of aminoacid numbers 20 to 470 of an amino acid sequence of SEQ ID NO: 44 and alight chain comprising an amino acid sequence consisting of amino acidnumbers 21 to 234 of the amino acid sequence of SEQ ID NO: 52, anantibody consisting of a heavy chain comprising an amino acid sequenceconsisting of amino acid numbers 20 to 470 of the amino acid sequence ofSEQ ID NO: 44 and a light chain comprising an amino acid sequenceconsisting of amino acid numbers 21 to 234 of the amino acid sequence ofSEQ ID NO: 54, an antibody consisting of a heavy chain comprising anamino acid sequence consisting of amino acid numbers 20 to 470 of theamino acid sequence of SEQ ID NO: 44 and a light chain comprising anamino acid sequence consisting of amino acid numbers 21 to 234 of theamino acid sequence of SEQ ID NO: 56, an antibody consisting of a heavychain comprising an amino acid sequence consisting of amino acid numbers20 to 470 of the amino acid sequence of SEQ ID NO: 46 and a light chaincomprising an amino acid sequence consisting of amino acid numbers 21 to234 of the amino acid sequence of SEQ ID NO: 54, an antibody consistingof a heavy chain comprising an amino acid sequence consisting of aminoacid numbers 20 to 470 of the amino acid sequence of SEQ ID NO: 46 and alight chain comprising an amino acid sequence consisting of amino acidnumbers 21 to 234 of the amino acid sequence of SEQ ID NO: 56, anantibody consisting of a heavy chain comprising an amino acid sequenceconsisting of amino acid numbers 20 to 470 of the amino acid sequence ofSEQ ID NO: 46 and a light chain comprising an amino acid sequenceconsisting of amino acid numbers 21 to 234 of the amino acid sequence ofSEQ ID NO: 58, an antibody consisting of a heavy chain comprising anamino acid sequence consisting of amino acid numbers 20 to 470 of theamino acid sequence of SEQ ID NO: 48 and a light chain comprising anamino acid sequence consisting of amino acid numbers 21 to 234 of theamino acid sequence of SEQ ID NO: 56, an antibody consisting of a heavychain comprising an amino acid sequence consisting of amino acid numbers20 to 470 of the amino acid sequence of SEQ ID NO: 107 and a light chaincomprising an amino acid sequence consisting of amino acid numbers 21 to234 of the amino acid sequence of SEQ ID NO: 52, and an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 470 of the amino acid sequence of SEQ ID NO:109 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 234 of the amino acid sequence of SEQ ID NO:56.

Further preferred examples of the combination can include an antibodyconsisting of a heavy chain comprising a heavy chain variable regionsequence consisting of amino acid numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 42 and a light chain comprising a light chainvariable region sequence consisting of amino acid numbers 21 to 129 ofthe amino acid sequence of SEQ ID NO: 52, an antibody consisting of aheavy chain comprising a heavy chain variable region sequence consistingof amino acid numbers 20 to 140 of the amino acid sequence of SEQ ID NO:44 and a light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 129 of the amino acid sequence ofSEQ ID NO: 56, an antibody consisting of a heavy chain comprising aheavy chain variable region sequence consisting of amino acid numbers 20to 140 of the amino acid sequence of SEQ ID NO: 107 and a light chaincomprising a light chain variable region sequence consisting of aminoacid numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 52, andan antibody consisting of a heavy chain comprising a heavy chainvariable region sequence consisting of amino acid numbers 20 to 140 ofthe amino acid sequence of SEQ ID NO: 109 and a light chain comprising alight chain variable region sequence consisting of amino acid numbers 21to 129 of the amino acid sequence of SEQ ID NO: 56.

Still further preferred examples of the combination can include anantibody consisting of a heavy chain comprising an amino acid sequenceconsisting of amino acid numbers 20 to 470 of the amino acid sequence ofSEQ ID NO: 42 and a light chain comprising an amino acid sequenceconsisting of amino acid numbers 21 to 234 of the amino acid sequence ofSEQ ID NO: 52, an antibody consisting of a heavy chain comprising anamino acid sequence consisting of amino acid numbers 20 to 470 of theamino acid sequence of SEQ ID NO: 44 and a light chain comprising anamino acid sequence consisting of amino acid numbers 21 to 234 of theamino acid sequence of SEQ ID NO: 56, an antibody consisting of a heavychain comprising an amino acid sequence consisting of amino acid numbers20 to 470 of the amino acid sequence of SEQ ID NO: 107 and a light chaincomprising an amino acid sequence consisting of amino acid numbers 21 to234 of the amino acid sequence of SEQ ID NO: 52, and an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 470 of the amino acid sequence of SEQ ID NO:109 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 234 of the amino acid sequence of SEQ ID NO:56.

Most preferred examples of the combination can include an antibodyconsisting of a heavy chain comprising an amino acid sequence consistingof amino acid numbers 20 to 470 of the amino acid sequence of SEQ ID NO:107 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 234 of the amino acid sequence of SEQ ID NO:52, and an antibody consisting of a heavy chain comprising an amino acidsequence consisting of amino acid numbers 20 to 470 of the amino acidsequence of SEQ ID NO: 109 and a light chain comprising an amino acidsequence consisting of amino acid numbers 21 to 234 of the amino acidsequence of SEQ ID NO: 56.

Actual examples of the humanized antibody derived from the A2-11Eantibody can include an arbitrary combination of a heavy chaincomprising a heavy chain variable region consisting of an amino acidsequence consisting of amino acid numbers 20 to 137 of the amino acidsequence of SEQ ID NO: 111 of the Sequence Listing or an amino acidsequence consisting of amino acid numbers 20 to 137 of the amino acidsequence of SEQ ID NO: 113, and a light chain comprising a light chainvariable region consisting of an amino acid sequence consisting of aminoacid numbers 21 to 128 of the amino acid sequence of SEQ ID NO: 115, anamino acid sequence consisting of amino acid numbers 21 to 128 of theamino acid sequence of SEQ ID NO: 117, or an amino acid sequenceconsisting of amino acid numbers 21 to 128 of the amino acid sequence ofSEQ ID NO: 119.

Preferred examples of the combination can include an antibody consistingof a heavy chain comprising a heavy chain variable region sequenceconsisting of amino acid numbers 20 to 137 of the amino acid sequence ofSEQ ID NO: 111 and a light chain comprising a light chain variableregion sequence consisting of amino acid numbers 21 to 128 of the aminoacid sequence of SEQ ID NO: 115, an antibody consisting of a heavy chaincomprising a heavy chain variable region sequence consisting of aminoacid numbers 20 to 137 of the amino acid sequence of SEQ ID NO: 111 anda light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 128 of the amino acid sequence ofSEQ ID NO: 117, an antibody consisting of a heavy chain comprising aheavy chain variable region sequence consisting of amino acid numbers 20to 137 of the amino acid sequence of SEQ ID NO: 111 and a light chaincomprising a light chain variable region sequence consisting of aminoacid numbers 21 to 128 of an amino acid sequence of SEQ ID NO: 119, anantibody consisting of a heavy chain comprising a heavy chain variableregion sequence consisting of amino acid numbers 20 to 137 of the aminoacid sequence of SEQ ID NO: 113 and a light chain comprising a lightchain variable region sequence consisting of amino acid numbers 21 to128 of the amino acid sequence of SEQ ID NO: 115, an antibody consistingof a heavy chain comprising a heavy chain variable region sequenceconsisting of amino acid numbers 20 to 137 of the amino acid sequence ofSEQ ID NO: 113 and a light chain comprising a light chain variableregion sequence consisting of amino acid numbers 21 to 128 of the aminoacid sequence of SEQ ID NO: 117, and an antibody consisting of a heavychain comprising a heavy chain variable region sequence consisting ofamino acid numbers 20 to 137 of the amino acid sequence of SEQ ID NO:113 and a light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 128 of the amino acid sequence ofSEQ ID NO: 119.

More preferred examples of the combination can include an antibodyconsisting of a heavy chain having an amino acid sequence consisting ofamino acid numbers 20 to 467 of the amino acid sequence of SEQ ID NO:111 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 233 of the amino acid sequence of SEQ ID NO:115, an antibody consisting of a heavy chain comprising an amino acidsequence consisting of amino acid numbers 20 to 467 of the amino acidsequence of SEQ ID NO: 111 and a light chain comprising an amino acidsequence consisting of amino acid numbers 21 to 233 of the amino acidsequence of SEQ ID NO: 117, an antibody consisting of a heavy chaincomprising an amino acid sequence consisting of amino acid numbers 20 to467 of the amino acid sequence of SEQ ID NO: 111 and a light chaincomprising an amino acid sequence consisting of amino acid numbers 21 to233 of the amino acid sequence of SEQ ID NO: 119, an antibody consistingof a heavy chain comprising an amino acid sequence consisting of aminoacid numbers 20 to 467 of an amino acid sequence of SEQ ID NO: 113 and alight chain comprising an amino acid sequence consisting of amino acidnumbers 21 to 233 of the amino acid sequence of SEQ ID NO: 115, anantibody consisting of a heavy chain comprising an amino acid sequenceconsisting of amino acid numbers 20 to 467 of the amino acid sequence ofSEQ ID NO: 113 and a light chain comprising an amino acid sequenceconsisting of amino acid numbers 21 to 233 of the amino acid sequence ofSEQ ID NO: 117, and an antibody consisting of a heavy chain comprisingan amino acid sequence consisting of amino acid numbers 20 to 467 of theamino acid sequence of SEQ ID NO: 113 and a light chain comprising anamino acid sequence consisting of amino acid numbers 21 to 233 of theamino acid sequence of SEQ ID NO: 119.

Actual examples of the humanized antibody derived from the A2-25Cantibody can include an arbitrary combination of a heavy chaincomprising a heavy chain variable region consisting of an amino acidsequence consisting of amino acid numbers 20 to 137 of the amino acidsequence of SEQ ID NO: 121 of the Sequence Listing or an amino acidsequence consisting of amino acid numbers 20 to 137 of the amino acidsequence of SEQ ID NO: 123, and a light chain comprising a light chainvariable region consisting of an amino acid sequence consisting of aminoacid numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 125, anamino acid sequence consisting of amino acid numbers 21 to 129 of theamino acid sequence of SEQ ID NO: 127, or an amino acid sequenceconsisting of amino acid numbers 21 to 129 of the amino acid sequence ofSEQ ID NO: 129.

Preferred examples of the combination can include an antibody consistingof a heavy chain comprising a heavy chain variable region sequenceconsisting of amino acid numbers 20 to 137 of the amino acid sequence ofSEQ ID NO: 121 and a light chain comprising a light chain variableregion sequence consisting of amino acid numbers 21 to 129 of the aminoacid sequence of SEQ ID NO: 125, an antibody consisting of a heavy chaincomprising a heavy chain variable region sequence consisting of aminoacid numbers 20 to 137 of the amino acid sequence of SEQ ID NO: 121 anda light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 129 of the amino acid sequence ofSEQ ID NO: 127, an antibody consisting of a heavy chain comprising aheavy chain variable region sequence consisting of amino acid numbers 20to 137 of the amino acid sequence of SEQ ID NO: 121 and a light chaincomprising a light chain variable region sequence consisting of aminoacid numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 129, anantibody consisting of a heavy chain comprising a heavy chain variableregion sequence consisting of amino acid numbers 20 to 137 of the aminoacid sequence of SEQ ID NO: 123 and a light chain comprising a lightchain variable region sequence consisting of amino acid numbers 21 to129 of the amino acid sequence of SEQ ID NO: 125, an antibody consistingof a heavy chain comprising a heavy chain variable region sequenceconsisting of amino acid numbers 20 to 137 of the amino acid sequence ofSEQ ID NO: 123 and a light chain comprising a light chain variableregion sequence consisting of amino acid numbers 21 to 129 of the aminoacid sequence of SEQ ID NO: 127, and an antibody consisting of a heavychain comprising a heavy chain variable region sequence consisting ofamino acid numbers 20 to 137 of the amino acid sequence of SEQ ID NO:123 and a light chain comprising a light chain variable region sequenceconsisting of amino acid numbers 21 to 129 of the amino acid sequence ofSEQ ID NO: 129.

More preferred examples of the combination can include an antibodyconsisting of a heavy chain having an amino acid sequence consisting ofamino acid numbers 20 to 467 of the amino acid sequence of SEQ ID NO:121 and a light chain comprising an amino acid sequence consisting ofamino acid numbers 21 to 234 of the amino acid sequence of SEQ ID NO:125, an antibody consisting of a heavy chain comprising an amino acidsequence consisting of amino acid numbers 20 to 467 of the amino acidsequence of SEQ ID NO: 121 and a light chain comprising an amino acidsequence consisting of amino acid numbers 21 to 234 of the amino acidsequence of SEQ ID NO: 127, an antibody consisting of a heavy chaincomprising an amino acid sequence consisting of amino acid numbers 20 to467 of the amino acid sequence of SEQ ID NO: 121 and a light chaincomprising an amino acid sequence consisting of amino acid numbers 21 to234 of the amino acid sequence of SEQ ID NO: 129, an antibody consistingof a heavy chain comprising an amino acid sequence consisting of aminoacid numbers 20 to 467 of the amino acid sequence of SEQ ID NO: 123 anda light chain comprising an amino acid sequence consisting of amino acidnumbers 21 to 234 of the amino acid sequence of SEQ ID NO: 125, anantibody consisting of a heavy chain comprising an amino acid sequenceconsisting of amino acid numbers 20 to 467 of the amino acid sequence ofSEQ ID NO: 123 and a light chain comprising an amino acid sequenceconsisting of amino acid numbers 21 to 234 of the amino acid sequence ofSEQ ID NO: 127, and an antibody consisting of a heavy chain comprisingan amino acid sequence consisting of amino acid numbers 20 to 467 of theamino acid sequence of SEQ ID NO: 123 and a light chain comprising anamino acid sequence consisting of amino acid numbers 21 to 234 of theamino acid sequence of SEQ ID NO: 129.

Further examples of the antibody of the present invention can include ahuman antibody. The anti-ALK2 human antibody means a human antibodyproduced from only human chromosome-derived antibody gene sequences. Theanti-ALK2 human antibody can be obtained by a method using humanantibody-producing mice carrying human chromosome fragments thatcomprise human antibody heavy and light chain genes (see e.g., Tomizuka,K. et al., Nature Genetics (1997), 16, p. 133-143; Kuroiwa, Y. et al.,Nuc. Acids Res. (1998), 26, p. 3447-3448; Yoshida, H. et al., AnimalCell Technology: Basic and Applied Aspects vol. 10, p. 69-73 (Kitagawa,Y., Matsuda, T. and ijima, S. eds.), Kluwer Academic Publishers, 1999;and Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, p.722-727).

Specifically, such a human antibody-producing mouse can be created as arecombinant animal in which the endogenous immunoglobulin heavy andlight chain gene loci have been disrupted and instead humanimmunoglobulin heavy and light chain gene loci are integrated via avector such as a human artificial chromosome (HAC) vector or a mouseartificial chromosome (MAC) vector, by preparing a knockout animal or atransgenic animal or by crossing these animals.

Alternatively, eukaryotic cells may be transformed with cDNAs encodingthe heavy and light chains, respectively, of such a human antibody,preferably with vectors comprising the cDNAs, by a gene recombinationtechnique. The transformed cells producing a recombinant humanmonoclonal antibody are cultured. This antibody can be obtained from theculture supernatant. In this context, for example, eukaryotic cells,preferably mammalian cells such as CHO cells, lymphocytes, or myelomacells, can be used as hosts.

Also, a method for obtaining a phage display-derived human antibodyselected from a human antibody library (see e.g., Wormstone, I. M. etal., Investigative Ophthalmology & Visual Science (2002), 43 (7), p.2301-2308; Carmen, S. et al., Briefings in Functional Genomics andProteomics (2002), 1 (2), p. 189-203; and Siriwardena, D. et al.,Ophthalmology (2002), 109 (3), p. 427-431) is known.

For example, a phage display method (Nature Biotechnology (2005), 23,(9), p. 1105-1116) can be used, which involves allowing the variableregions of a human antibody to be expressed as single-chain Fv (scFv) onphage surface and selecting a phage binding to the antigen. The phageselected on the basis of its ability to bind to the antigen can besubjected to gene analysis to determine DNA sequences encoding thevariable regions of the human antibody binding to the antigen. If theDNA sequence of scFv binding to the antigen is determined, an expressionvector having this sequence can be prepared and transferred toappropriate hosts, followed by expression to obtain the human antibody(WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438,WO95/15388, Annu. Rev. Immunol (1994), 12, p. 433-455; and NatureBiotechnology (2005), 23 (9), p. 1105-1116).

Antibodies binding to the same epitope as that for the antibody providedby the present invention are also included in the antibodies of thepresent invention. Examples thereof include antibodies binding to thesame epitope as that for the A2-11E antibody, the A2-15A antibody, theA2-25C antibody, and/or the A2-27D antibody.

When an antibody binds to or recognizes a partial conformation of anantigen, the epitope for this antibody can be determined by identifyingamino acid residues on the antigen adjacent to the antibody by use ofX-ray structure analysis. For example, the antibody or a fragmentthereof and the antigen or a fragment thereof can be bound to eachother, crystallized, and structurally analyzed to identify amino acidresidues on the antigen having an interaction distance between the aminoacid residue and the antibody. The interaction distance is 8 angstromsor smaller, preferably 6 angstroms or smaller, more preferably 4angstroms or smaller. One or more amino acid residues having such aninteraction distance with the antibody can constitute the epitope(antigenic determinant) for the antibody. When the number of such aminoacid residues is two or more, these amino acids may not be adjacent toeach other on the primary sequence.

A Fab fragment of the chimeric A2-27D antibody and a peptide containingan ECD fragment (amino acid numbers 21 to 123 of SEQ ID NO: 84) of humanALK2 are bound to each other and crystallized under conditions involving2% (v/v) Tacsimate (pH 7.0), 100 mM HEPES (pH 7.5), and 20% (w/v)polyethylene glycol 3,350 to obtain crystals having a body-centeredmonoclinic crystal system with a space group C121 and unit cells ofa=c=119.39 angstroms, b=37.32 angstroms, and β=92.54. The phase can bedetermined by a molecular replacement method using three-dimensionalstructure coordinates thereof (see Example 16).

The A2-27D antibody recognizes a partial conformation on human ALK2. Inthe amino acid sequence (SEQ ID NO: 84) of human ALK2, the amino acidresidues having an interaction distance with the A2-27D antibody, i.e.,the epitope, is constituted by each of the residues of glutamic acid(Glu) at position 18, glycine (Gly) at position 19, isoleucine (Ile) atposition 39, asparagine (Asn) at position 40, aspartic acid (Asp) atposition 41, glycine (Gly) at position 42, phenylalanine (Phe) atposition 43, histidine (His) at position 44, valine (Val) at position45, tyrosine (Tyr) at position 46, asparagine (Asn) at position 82,threonine (Thr) at position 84, glutamine (Gln) at position 86, leucine(Leu) at position 87, proline (Pro) at position 88, and threonine (Thr)at position 89. The antibody, an antigen-binding fragment thereof, or amodified form of the antibody or the fragment which binds to thisepitope or has an interaction distance between the antibody or thefragment and each of the amino acid residues are also encompassed by theantibody of the present invention, the antigen-binding fragment thereof,or a modified form of the antibody or the fragment.

A Fab fragment of the chimeric A2-25C antibody and a peptide containingan ECD fragment (amino acid numbers 21 to 123 of SEQ ID NO: 84) of humanALK2 are bound to each other and crystallized under conditions involving0.15 M Li₂SO₄, 0.1 M Na citrate (pH 3.4), 18% (w/v) PEG6,000, and 20%(v/v) ethylene glycol to obtain crystals having an orthorhombic crystalsystem with a space group P2₁2₁2₁ and unit cells of a=74.49 angstroms,b=128.05 angstroms, and c=147.73 angstroms. The phase can be determinedby a molecular replacement method using three-dimensional structurecoordinates thereof (see Example 21).

The A2-25C antibody recognizes a partial conformation on human ALK2. Inthe amino acid sequence (SEQ ID NO: 84) of human ALK2, the amino acidresidues having an interaction distance with the A2-25C antibody, i.e.,the epitope, is constituted by each of the residues of glutamic acid(Glu) at position 18, glycine (Gly) at position 19, leucine (Leu) atposition 20, isoleucine (Ile) at position 39, asparagine (Asn) atposition 40, aspartic acid (Asp) at position 41, glycine (Gly) atposition 42, phenylalanine (Phe) at position 43, histidine (His) atposition 44, valine (Val) at position 45, tyrosine (Tyr) at position 46,and threonine (Thr) at position 84. The antibody, an antigen-bindingfragment thereof, or a modified form of the antibody or the fragmentwhich binds to this epitope or has an interaction distance with theseamino acid residues are also encompassed by the antibody of the presentinvention, the antigen-binding fragment thereof, or a modified form ofthe antibody or the fragment.

The antibody described above can be evaluated for its binding activityto the antigen by, for example, a method described in Example 2, 6, 9,or 10 to select suitable antibodies. The dissociation constant of theantibody is, for example, 1×10⁻⁶ to 1×10⁻¹² M, but is not limited tothis range as long as the antibody of the present invention produces thetherapeutic or prophylactic effects of interest. The dissociationconstant of the antibody for the antigen (ALK2) can be measured usingBiacore T200 (GE Healthcare Bio-Sciences Corp.) based on surface plasmonresonance (SPR) as detection principles. For example, the antibody setto an appropriate concentration is reacted as an analyte with theantigen immobilized as a ligand on a solid phase. The association anddissociation therebetween can be measured to obtain an association rateconstant ka1, a dissociation rate constant kd1, and a dissociationconstant (KD; KD=kd1/ka1). The evaluation of binding activity againstALK2 is not limited by use of Biacore T200 and may be conducted using,for example, an instrument based on surface plasmon resonance (SPR) asdetection principles, KinExA (Sapidyne Instruments Inc.) based onkinetic exclusion assay as detection principles, BLItz system (PallCorp.) based on bio-layer interferometry as detection principles, orELISA (enzyme-linked immunosorbent assay).

One example of another index for comparing the properties of antibodiescan include the stability of the antibodies. Differential scanningcalorimetry (DSC) is a method that can rapidly and accurately measure atransition midpoint (Tm), which serves as a good index for the relativestructural stability of proteins. Tm values can be measured using DSCand compared to determine difference in thermal stability. Thepreservation stability of an antibody is known to correlate with thethermal stability of the antibody to some extent (Lori Burton, et al.,Pharmaceutical Development and Technology (2007) 12, p. 265-273). Asuitable antibody can be selected with its thermal stability as theindex. Examples of other indexes for selecting the antibody can includehigh yields in appropriate host cells and low aggregation in an aqueoussolution. For example, an antibody having the highest yield does notalways exhibit the highest thermal stability. Therefore, it is necessaryto select an antibody most suitable for administration to humans bycomprehensive judgment based on the indexes mentioned above.

A method for obtaining a single-chain immunoglobulin by linking thefull-length sequences of antibody heavy and light chains using anappropriate linker is also known (Lee, H-S, et al., Molecular Immunology(1999) 36, p. 61-71; and Shirrmann, T. et al., mAbs (2010), 2, (1) p.1-4). Such single-chain immunoglobulins can be dimerized to retain astructure and activity similar to those of antibodies which areoriginally tetramers. Alternatively, the antibody of the presentinvention may be an antibody that has a single heavy chain variableregion and lacks a light chain sequence. Such an antibody, calledsingle-domain antibody (sdAb) or nanobody, has actually been observed incamels or llamas and has been reported to maintain the ability to bindto an antigen (Muyldemans S. et al., Protein Eng. (1994) 7 (9), 1129-35;and Hamers-Casterman C. et al., Nature (1993) 363 (6428) 446-8). Theseantibodies may also be interpreted as an antigen-binding fragment of theantibody according to the present invention.

The antibody-dependent cellular cytotoxic activity of the antibody ofthe present invention can be enhanced by adjusting the modification ofthe sugar chain bound with the antibody. For example, methods describedin WO99/54342, WO2000/61739, and WO2002/31140 are known as such atechnique of adjusting the sugar chain modification of the antibody,though this technique is not limited thereto.

In the case of preparing an antibody by temporarily isolating theantibody gene and then transferring the gene to an appropriate host, theappropriate host can be used in combination with an expression vector.

Specific examples of the antibody gene can include a gene (or apolynucleotide) encoding a heavy chain sequence and a gene (or apolynucleotide) encoding a light chain sequence of the antibodydescribed herein, and a combination of these genes (or polynucleotides).

Specific examples of the polynucleotide encoding the antibody or anantibody constituent (heavy or light chain) are as follows.

(1) A polynucleotide comprising the following polynucleotides (a) and/or(b):

(a) a polynucleotide selected from the group consisting of the followingnucleotide sequences:a1) a nucleotide sequence consisting of nucleotide numbers 58 to 426 ofthe nucleotide sequence of SEQ ID NO: 27;a2) a nucleotide sequence consisting of nucleotide numbers 58 to 426 ofthe nucleotide sequence of SEQ ID NO: 29;a3) a nucleotide sequence consisting of nucleotide numbers 58 to 426 ofthe nucleotide sequence of SEQ ID NO: 104;a4) a nucleotide sequence having at least 95% identity to any of thenucleotide sequences a1) to a3);a5) a nucleotide sequence having at least 99% identity to any of thenucleotide sequences a1) to a3);a6) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any of the nucleotide sequences a1) to a3);anda7) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in the nucleotidesequence a1) or a2); and/or(b) a polynucleotide selected from the group consisting of the followingnucleotide sequences:b1) a nucleotide sequence consisting of nucleotide numbers 86 to 424 ofthe nucleotide sequence of SEQ ID NO: 31;b2) a nucleotide sequence consisting of nucleotide numbers 86 to 424 ofthe nucleotide sequence of SEQ ID NO: 33;b3) a nucleotide sequence consisting of nucleotide numbers 86 to 424 ofthe nucleotide sequence of SEQ ID NO: 35;b4) a nucleotide sequence consisting of nucleotide numbers 86 to 424 ofthe nucleotide sequence of SEQ ID NO: 37;b5) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b4);b6) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b4);b7) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences b1) to b4); andb8) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequencesb1) to b4).

(2) A polynucleotide comprising the following polynucleotides (a) and/or(b):

(a) a polynucleotide selected from the group consisting of the followingnucleotide sequences:a1) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 39;a2) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 41;a3) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 43;a4) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 45;a5) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 47;a6) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 106;a7) a nucleotide sequence consisting of nucleotide numbers 58 to 420 ofthe nucleotide sequence of SEQ ID NO: 108;a8) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences a1) to a7);a9) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences a1) to a7);a10) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences a1) to a7); anda11) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequences a1) to a7); and/or(b) a polynucleotide selected from the group consisting of the followingnucleotide sequences:b1) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 49;b2) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 51;b3) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 53;b4) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 55;b5) a nucleotide sequence consisting of nucleotide numbers 86 to 412 ofthe nucleotide sequence of SEQ ID NO: 57;b6) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b5);b7) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b5);b8) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences b1) to b5); andb9) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequencesb1) to b5).

(3) A polynucleotide comprising the following polynucleotides (a) and/or(b):

(a) a polynucleotide selected from the group consisting of the followingnucleotide sequences:a1) a nucleotide sequence consisting of nucleotide numbers 58 to 411 ofthe nucleotide sequence of SEQ ID NO: 110;a2) a nucleotide sequence consisting of nucleotide numbers 58 to 411 ofthe nucleotide sequence of SEQ ID NO: 112;a3) a nucleotide sequence having at least 95% identity to the nucleotidesequence a1) or a2);a4) a nucleotide sequence having at least 99% identity to the nucleotidesequence a1) or a2);a5) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to the nucleotide sequence a1) or a2); anda6) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in the nucleotidesequence a1) or a2); and/or(b) a polynucleotide selected from the group consisting of the followingnucleotide sequences:b1) a nucleotide sequence consisting of nucleotide numbers 61 to 384 ofthe nucleotide sequence of SEQ ID NO: 114;b2) a nucleotide sequence consisting of nucleotide numbers 61 to 384 ofthe nucleotide sequence of SEQ ID NO: 116;b3) a nucleotide sequence consisting of nucleotide numbers 61 to 384 ofthe nucleotide sequence of SEQ ID NO: 118;b4) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b3);b5) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b3);b6) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences b1) to b3); andb7) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequencesb1) to b3).

(4) A polynucleotide comprising the following polynucleotides (a) and/or(b):

(a) a polynucleotide selected from the group consisting of the followingnucleotide sequences:a1) a nucleotide sequence consisting of nucleotide numbers 58 to 411 ofthe nucleotide sequence of SEQ ID NO: 120;a2) a nucleotide sequence consisting of nucleotide numbers 58 to 411 ofthe nucleotide sequence of SEQ ID NO: 122;a3) a nucleotide sequence having at least 95% identity to the nucleotidesequence a1) or a2);a4) a nucleotide sequence having at least 99% identity to the nucleotidesequence a1) or a2);a5) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to the nucleotide sequence a1) or a2); anda6) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in the nucleotidesequence a1) or a2); and/or(b) a polynucleotide selected from the group consisting of the followingnucleotide sequences:b1) a nucleotide sequence consisting of nucleotide numbers 61 to 387 ofthe nucleotide sequence of SEQ ID NO: 124;b2) a nucleotide sequence consisting of nucleotide numbers 61 to 387 ofthe nucleotide sequence of SEQ ID NO: 126;b3) a nucleotide sequence consisting of nucleotide numbers 61 to 387 ofthe nucleotide sequence of SEQ ID NO: 128;b4) a nucleotide sequence having at least 95% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b3);b5) a nucleotide sequence having at least 99% identity to any onenucleotide sequence selected from the nucleotide sequences b1) to b3);b6) a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences b1) to b3); andb7) a nucleotide sequence comprising a substitution(s), a deletion(s),or an addition(s) of one to several nucleotides in any one nucleotidesequence selected from the nucleotide sequencesb1) to b3).

For the transformation of host cells, a heavy chain sequence gene (orpolynucleotide) and a light chain sequence gene (or polynucleotide) maybe inserted in a same expression vector or may be inserted in separateexpression vectors. In the case of using host eukaryotic cells, animalcells, plant cells, or eukaryotic microorganisms can be used. Examplesof the animal cells can include mammalian cells, for example, monkey COScells (Gluzman, Y., Cell (1981) 23, p. 175-182, ATCC CRL-1650), mousefibroblast NIH3T3 (ATCC No. CRL-1658), and dihydrofolatereductase-deficient cell lines (Urlaub, G. and Chasin, L. A., Proc.Natl. Acad. Sci. U.S.A. (1980) 77, p. 4126-4220) of Chinese hamsterovary cells (CHO cells, ATCC CCL-61). In the case of using prokaryoticcells, examples thereof can include E. coli and Bacillus subtilis. Theantibody gene of interest is transferred to these cells bytransformation, and the transformed cells are cultured in vitro toobtain antibodies. Such culture methods may differ in yield depending onthe sequences of the antibodies. An antibody that is easy to produce asa drug can be selected with its yield as an index from among antibodieshaving equivalent binding activity.

Examples of the isotype of the antibody of the present invention caninclude, but are not limited to, IgG (IgG1, IgG2, IgG3, and IgG4), IgM,IgA (IgA1 and IgA2), IgD, and IgE. Preferred examples of the isotype caninclude IgG and IgM, more preferably IgG1, IgG2, and IgG4.

In the case of using IgG1 as an isotype of the antibody of the presentinvention, the effector functions can be adjusted by substitution of apart of amino acid residues in constant regions (see WO88/07089,WO94/28027, and WO94/29351). Examples of such a variant of IgG1 includeIgG1 LALA (IgG1-L234A, L235A) and IgG1 LAGA (IgG1-L235A, G237A). IgG1LALA is preferred.

In the case of using IgG4 as an isotype of the antibody of the presentinvention, splitting unique to IgG4 can be suppressed with extendedhalf-life by substitution of a part of amino acid residues in constantregions (see Molecular Immunology, 30, 1 105-108 (1993)). Examples ofsuch a mutant of IgG4 include IgG4 pro (IgG4-S241P).

The antibody of the present invention may be an antigen-binding fragmentof the antibody having the antigen-binding site of the antibody, or amodified form thereof. A fragment of the antibody can be obtained bytreating the antibody with a proteolytic enzyme such as papain or pepsinor by expressing a genetically engineered antibody gene in appropriatecultured cells. Among such antibody fragments, a fragment that maintainsthe whole or a portion of the functions possessed by the full-lengthmolecule of the antibody can be referred to as an antigen-bindingfragment of the antibody. Examples of the functions of the antibody cangenerally include antigen binding activity, activity of inhibiting theactivity of the antigen, activity of enhancing the activity of theantigen, antibody-dependent cellular cytotoxic activity,complement-dependent cytotoxic activity, and complement-dependentcellular cytotoxic activity. The function possessed by theantigen-binding fragment of the antibody according to the presentinvention is an activity of binding to ALK2 and is preferably anactivity of inhibiting the activity of ALK2, more preferably an activityof inhibiting ALK2-mediated BMP signal transduction, most preferably anactivity of suppressing ectopic ossification and/or bone dysplasia.

Examples of the fragment of the antibody can include Fab, F(ab′)2, Fv,single-chain Fv (scFv) comprising heavy and light chain Fvs linked viaan appropriate linker, diabody or diabodies, linear antibodies, andmultispecific antibodies formed from antibody fragments. The fragment ofthe antibody also includes Fab′, which is a monovalent fragment ofantibody variable regions obtained by the treatment of F(ab′)2 underreducing conditions.

The antibody of the present invention may be a multispecific antibodyhaving specificity for at least two different types of antigens. Such amolecule usually binds to two types of antigens (i.e., a bispecificantibody). The “multispecific antibody” according to the presentinvention encompasses an antibody having specificity for more types(e.g., 3 types) of antigens.

The multispecific antibody of the present invention may be an antibodyconsisting of a full length, or may be a fragment of such an antibody(e.g., a bispecific antibody). The bispecific antibody may be preparedby linking the heavy and light chains (HL pairs) of two types ofantibodies, or may be prepared by fusing hybridomas producing differentmonoclonal antibodies to prepare bispecific antibody-producing fusioncells (Millstein et al., Nature (1983) 305, p. 537-539).

The antibody of the present invention may be single-chain Fv (alsoreferred to as scFv). The single-chain Fv is obtained by linking theheavy and light chain variable regions of the antibody via a polypeptidelinker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113(Rosenberg and Moore ed., Springer Verlag, New York, p. 269-315 (1994);and Nature Biotechnology (2005), 23, p. 1126-1136). Also, a biscFvfragment prepared by linking two scFvs via a polypeptide linker can alsobe used as a bispecific antibody.

The method for preparing the single-chain Fv is well-known in the art(see e.g., U.S. Pat. Nos. 4,946,778, 5,260,203, 5,091,513, and5,455,030). In this scFv, the heavy chain variable region and the lightchain variable region are linked via a linker that prevents theseregions from forming a conjugate, preferably a polypeptide linker(Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988), 85, p.5879-5883). The heavy chain variable region and the light chain variableregion in the scFv may be derived from the same antibody or may bederived from different antibodies. For example, an arbitrarysingle-chain peptide consisting of 12 to 19 residues is used as thepolypeptide linker that links these variable regions.

In order to obtain DNA encoding the scFv, each DNA portion encoding thewhole or desired amino acid sequence in the sequences of DNA encodingthe heavy chain or heavy chain variable region of the antibody and DNAencoding the light chain or light chain variable region thereof, is usedas a template and amplified by PCR using a primer pair flanking bothends of the template. Subsequently, DNA encoding the polypeptide linkermoiety is further amplified in combination with a primer pair defined sothat the polypeptide linker moiety can be linked at its ends to theheavy and light chain DNAs, respectively.

Once the DNA encoding the scFv is prepared, an expression vectorcomprising the DNA and a host transformed with the expression vector canbe obtained by routine methods. In addition, the host can be used toobtain the scFv according to a routine method. These antibody fragmentscan be produced by a host in the same way as above by obtaining andexpressing the gene.

The antibody of the present invention may have enhanced affinity for theantigen by multimerization. Antibodies of the same type may bemultimerized, or a plurality of antibodies recognizing a plurality ofepitopes, respectively, of the same antigen may be multimerized.Examples of a method for multimerizing these antibodies can include thebinding of two scFvs to an IgG CH3 domain, the binding thereof tostreptavidin, and the introduction of a helix-turn-helix motif.

The antibody of the present invention may be a polyclonal antibody whichis a mixture of plural types of anti-ALK2 antibodies differing in aminoacid sequence. One example of the polyclonal antibody can include amixture of plural types of antibodies differing in CDRs. An antibodyobtained by culturing a mixture of cells producing different antibodies,followed by purification from the cultures can be used as such apolyclonal antibody (see WO2004/061104).

The antibody of the present invention may be an antibody having 80% to99% identity as compared with the heavy and/or light chains of theantibody. In this context, the term “identity” has general definitionused in the art. The % identity refers to the percentage of the numberof identical amino acids relative to the total number of amino acids(including gaps) when two amino acid sequences are aligned so as to givethe largest consistency of amino acids. Antibodies that have an abilityto bind to the antigen and an inhibitory effect on BMP signaltransduction at analogous levels to the antibodies described above canbe selected by combining sequences that exhibit high identity to theamino acid sequences of the heavy and light chains. Such identity isgenerally 80% or higher identity, preferably 90% or higher identity,more preferably 95% or higher identity, most preferably 99% or higheridentity. Alternatively, antibodies that have various effects equivalentto the antibodies described above may be selected by combining aminoacid sequences that comprise a substitution(s), a deletion(s), and/or anaddition(s) of one to several amino acid residues in the amino acidsequences of the heavy and/or light chains. The number of amino acidresidues to be substituted, deleted, and/or added is generally 10 orless amino acid residues, preferably 5 or 6 or less amino acid residues,more preferably 2 or 3 or less amino acid residues, most preferably 1amino acid residue.

The heavy chain of an antibody produced by cultured mammalian cells isknown to lack a carboxyl-terminal lysine residue (Journal ofChromatography A, 705: 129-134 (1995)). Also, the heavy chain of such anantibody is known to lack two carboxyl-terminal amino acid residues(glycine and lysine) and instead have an amidated proline residue at thecarboxy terminus (Analytical Biochemistry, 360: 75-83 (2007)).

An amino-terminal glutamine or glutamic acid residue in the heavy orlight chain of an antibody is known to be modified by pyroglutamylationduring preparation of the antibody, and the antibody of the presentinvention may have such a modification (WO2013/147153).

Such deletion in the heavy chain sequence or modification in the heavyor light chain sequence does not influence the ability of the antibodyto bind to the antigen and its effector functions (complementactivation, antibody-dependent cytotoxic effects, etc.).

Thus, the present invention also encompasses an antibody that hasreceived the deletion or the modification. Examples thereof can includea deletion variant derived from a heavy chain by the deletion of 1 or 2amino acids at its carboxyl terminus, an amidated form of the deletionvariant (e.g., a heavy chain having an amidated proline residue at thecarboxyl-terminal site), and an antibody having a pyroglutamylatedamino-terminal amino acid residue in a heavy or light chain thereof.However, the deletion variant at the carboxyl terminus of the antibodyheavy chain according to the present invention is not limited to thetypes described above as long as the deletion variant maintains theability to bind to the antigen and the effector functions. Two heavychains constituting the antibody according to the present invention maybe heavy chains of any one type selected from the group consisting ofthe full-length heavy chain and the deletion variants described above,or may be a combination of heavy chains of any two types selectedtherefrom. The quantitative ratio of each deletion variant may beinfluenced by the type of cultured mammalian cells producing theantibody according to the present invention, and culture conditions.Examples of such a case can include the deletion of onecarboxyl-terminal amino acid residue each in both the two heavy chainsas main components of the antibody according to the present invention.

The identity between two types of amino acid sequences can be determinedusing the default parameters of Blast algorithm version 2.2.2 (Altschul,Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang,Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25: 3389-3402). The Blast algorithm is also availableby access to www.ncbi.nlm.nih.gov/blast on the Internet. Two types ofpercentage values, Identity (or Identities) and Positivity (orPositivities), are calculated according to the Blast algorithm. Theformer is a value that indicates identical amino acid residues betweentwo types of amino acid sequences between which the identity should bedetermined. The latter is a numerical value determined by also takinginto consideration similar amino acid residues in terms of theirchemical structures. Herein, the value of identity is defined as thevalue of “Identity” when amino acid residues are identical between theamino acid sequences.

An antibody conjugated with any of various molecules such aspolyethylene glycol (PEG) can also be used as a modified form of theantibody.

The antibody of the present invention may further be any of conjugatesformed by these antibodies with other drugs (immunoconjugates). Examplesof such an antibody can include the antibody conjugated with aradioactive material or a compound having a pharmacological effect(Nature Biotechnology (2005) 23, p. 1137-1146).

The obtained antibody can be purified until homogeneous. A proteinseparation and purification method usually used can be used for theseparation and purification of the antibody. The antibody can beseparated and purified by appropriately selected or combined approaches,for example, column chromatography, filtration through a filter,ultrafiltration, salting-out, dialysis, preparative polyacrylamide gelelectrophoresis, and/or isoelectric focusing (Strategies for ProteinPurification and Characterization: A Laboratory Course Manual, Daniel R.Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); andAntibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold SpringHarbor Laboratory (1988)), though the separation and purification methodis not limited thereto.

Examples of the chromatography can include affinity chromatography,ion-exchange chromatography, hydrophobic chromatography, gel filtrationchromatography, reverse-phase chromatography, and adsorptionchromatography.

These chromatography approaches can be carried out using liquidchromatography such as HPLC or FPLC.

Examples of the column for use in the affinity chromatography caninclude protein A columns and protein G columns.

Examples of the protein A columns can include Hyper D, POROS, andSepharose F. F. (GE Healthcare Bio-Sciences Corp.).

Also, the antibody may be purified by exploiting its binding activity tothe antigen using an antigen-immobilized carrier.

5. Drug Comprising Anti-ALK2 Antibody

An antibody inhibiting the biological activity of ALK2 can be obtainedfrom among anti-ALK2 antibodies obtained by the methods described abovein the preceding paragraph “4. Production of anti-ALK2 antibody”. Suchan antibody inhibiting the biological activity of ALK2 inhibits thebiological activity of ALK2, i.e., ALK2-mediated BMP signaltransduction, in vivo and as such, can be pharmaceutically used as atherapeutic and/or prophylactic drug for ectopic ossification and/orbone dysplasia or a therapeutic and/or prophylactic drug for anemia.

Examples of the disease that can be treated and/or prevented with theanti-ALK2 antibody can include fibrodysplasia ossificans progressiva(FOP), progressive osseous heteroplasia (POH), traumatic ectopicossification, ectopic ossification after implant arthroplasty,spondyloarthritis (SpA), ankylosing spondylitis (AS), anemia, diffuseintrinsic pontine glioma (DIPG), and thinning hair. The disease ispreferably fibrodysplasia ossificans progressiva (FOP), progressiveosseous heteroplasia (POH), traumatic ectopic ossification, or ectopicossification after implant arthroplasty, more preferably fibrodysplasiaossificans progressiva (FOP), though the disease is not limited theretoas long as the disease is caused by ALK2-mediated BMP signaltransduction. In FOP patients, finger or toe fusion or deformity,cervical fusion or deformity, or the like is also found, and hearingloss is also manifested. These conditions are also included in thedisease caused by ALK2-mediated BMP signal transduction.

Activating mutations in ALK2 have been confirmed in all FOP patients,and 10 or more types of mutations have been reported so far. All ofthese mutations have been found to be amino acid mutations (missensemutations) present in the intracellular region of the ALK2 protein anddo not cause any change in the amino acid sequence of the extracellularregion. The antibody of the present invention binds to the extracellularregion of ALK2 and as such, can be used as a therapeutic and/orprophylactic drug for every FOP patient, irrespective of the types ofmutations.

The treatment of FOP means cure of FOP symptoms, amelioration of thesymptoms, mitigation of the symptoms, or suppression of progression ofthe symptoms.

The prevention of FOP means circumvention or suppression of onset offlare-up or ectopic ossification.

The antibody of the present invention also binds not only to mutatedALK2 but to wild-type ALK2 and inhibits downstream signals. Therefore,the antibody of the present invention can also be used as a therapeuticand/or prophylactic drug for non-hereditary ectopic ossificationdifferent from FOP. Examples of the non-hereditary ectopic ossificationinclude ectopic ossification after brain contusion, ectopic ossificationafter spinal cord injury, ectopic ossification after burn injury, andectopic ossification after implant arthroplasty. The involvement ofneuropeptides, fat cells, and immune system cells such as macrophageshas been reported as a cause for the non-hereditary ectopic ossification(see J. Cell. Biochem., 112, 10 (2011); J. Cell. Biochem., 112, 10(2011); and J. Pathol., 236, 2 (2015)). It has been suggested that asimilar mechanism is also involved in flare-up in FOP patients (see Hum.Pathol., 32, 8 (2001); and Histol. Histopathol., 29, 10 (2014)). Thus,the antibody of the present invention can be used as a therapeuticand/or prophylactic drug not only for ectopic ossification in FOP butfor non-hereditary ectopic ossification.

Hepcidin gene is known as a target gene whose transcription ispositively regulated by the BMP-ALK signal transduction pathway inanemia (see Blood, 118, 15 (2011)). Hepcidin is a peptide hormone mainlyproduced in the liver, and regulates the degradation of a transporter,called ferroportin, which is expressed in the gastrointestinal tract andinvolved in iron absorption (see Science, 306, 5704 (2004)). A potentialtherapeutic drug for anemia has been reported because the functionalinhibition of hepcidin or the suppression of its expression level leadsto the promotion of iron uptake from the gastrointestinal tract viaincrease in the expression level of ferroportin (see Pharmacol Ther, 146(2015)). Thus, the antibody of the present invention can also be used asa therapeutic and/or prophylactic drug for iron-deficiency anemia byincreasing iron absorption via the suppression of hepcidin expression inthe liver.

Alternatively, the antibody of the present invention may be used as atherapeutic and/or prophylactic drug for diffuse intrinsic pontineglioma (DIPG). DIPG is diffuse (infiltrative) astrocytoma that is foundmainly in the pons of the brain stem and reportedly accounts forapproximately 75 to 80% of pediatric brain stem tumors. There is areport stating that fewer than about 10% of children with DIPG survivefor more than 2 years, because the brain stem regulates essentialfunctions such as respiration (Khuong-Quang D-A et al., Acta Neuropathol124: 439-447, 2012). The antibody of the present invention can also beused as a therapeutic and/or prophylactic drug for DIPG.

Examples of the anti-ALK2 antibody as these drugs can include chimericantibodies and humanized antibodies prepared by the methods described in“4. Production of anti-ALK2 antibody” from the A2-15A antibody, theA2-27D antibody, the A2-11E antibody, or the A2-25C antibody. A chimericantibody, a humanized antibody and a human antibody binding to the sameepitope as that for the A2-15A antibody, the A2-27D antibody, the A2-11Eantibody, and/or the A2-25C antibody may also be used as the drugs.

The biological activity of ALK2 (BMP signal inhibitory activity) of theanti-ALK2 antibody can be confirmed in vitro, for example, by luciferaseassay using reporter plasmids having an insert of a BMP-responsivesequence, SMAD1/5/8 phosphorylation, expression analysis of BMP targetgenes, or measurement of alkaline phosphatase activity in mousemyoblasts C2C12 induced to differentiate into osteoblasts by stimulationwith a BMP ligand.

The therapeutic or prophylactic effects of the anti-ALK2 antibody onectopic ossification or bone dysplasia can be confirmed in vivo usinglaboratory animals, for example, by subcutaneously or intravenouslyadministering the anti-ALK2 antibody to ectopic ossification-inducedmodels with BMP ligand-containing pellets transplanted to mouse muscle,or FOP mouse models harboring mutated ALK2, and analyzing ectopic boneformation.

The anti-AKL2 antibody thus obtained is useful as a drug, particularly,a pharmaceutical composition aimed at treating or preventing ectopicossification such as fibrodysplasia ossificans progressiva (FOP).

As one example, the anti-ALK2 antibody can be administered alone or incombination with at least one additional therapeutic drug for ectopicossification in the treatment or prevention of ectopic ossification. Asanother example, the anti-ALK2 antibody can be administered incombination with a therapeutically effective amount of a therapeuticdrug. Examples of the additional therapeutic drug for ectopicossification that can be administered in combination with the anti-ALK2antibody can include, but are not limited to, anti-inflammatory drugs,steroids, bisphosphonates, muscle relaxants, and retinoic acid receptor(RAR) γ agonists.

Examples of the anti-inflammatory drug can include aspirin, diclofenac,indomethacin, ibuprofen, ketoprofen, naproxen, piroxicam, rofecoxib,celecoxib, azathioprine, penicillamine, methotrexate, sulfasalazine,leflunomide, infliximab, and etanercept. Indomethacin, ibuprofen,piroxicam, or celecoxib is preferred.

Examples of the steroid can include prednisolone, beclomethasone,betamethasone, fluticasone, dexamethasone, and hydrocortisone.Prednisolone is preferred.

Examples of the bisphosphonate can include alendronate, cimadronate,clodronate, etidronate, ibandronate, incadronate, minodronate,neridronate, olpadronate, pamidronate, piridronate, risedronate,tiludronate, and zoledronate. Pamidronate or zoledronate is preferred.

Examples of the muscle relaxant can include cyclobenzaprine, metaxalone,and baclofen. Baclofen is preferred.

Examples of the retinoic acid receptor γ agonist can includepalovarotene.

Depending on the condition of ectopic ossification or the intendeddegree of treatment and/or prevention, two or three or more additionaltherapeutic drugs can be administered, and these additional therapeuticdrugs can be included in the same preparation and thereby administeredat the same time. The additional therapeutic drug and the anti-ALK2antibody can also be included in the same preparation and therebyadministered at the same time. Also, the anti-ALK2 antibody and theadditional therapeutic drug can be included in separate preparations andadministered at the same time. Alternatively, the additional agent andthe anti-ALK2 antibody may be separately administered one after another.Specifically, a therapeutic drug comprising the anti-ALK2 antibody orthe antigen-binding fragment of the antibody as an active ingredient maybe administered after administration of the additional therapeutic drug,or the additional therapeutic drug may be administered afteradministration of the therapeutic drug containing the anti-ALK2 antibodyor the antigen-binding fragment of the antibody as an active ingredient.For administration in gene therapy, a gene for a protein serving as atherapeutic drug for ectopic ossification and the gene for the anti-ALK2antibody can be inserted at a site downstream of separate promoterregions or the same promoter region and can be introduced to separatevectors or the same vector.

The anti-ALK2 antibody or the fragment thereof can be conjugated with atherapeutic drug for ectopic ossification to produce a targeted drugconjugate described in M. C. Garnet “Targeted drug conjugates:principles and progress”, Advanced Drug Delivery Reviews, (2001) 53,171-216. For this purpose, an antibody molecule as well as any antibodyfragment is applicable unless their ALK2-recognizing properties arecompletely deleted. Examples of the antibody fragment can includefragments such as Fab, F(ab′)2, and Fv. In the present invention aswell, the antibody and the fragment can be used. The conjugation mannerof the anti-ALK2 antibody or the fragment of the antibody with thetherapeutic drug for FOP can take various forms described in, forexample, M. C. Garnet “Targeted drug conjugates: principles andprogress”, Advanced Drug Delivery Reviews, (2001) 53, 171-216, G. T.Hermanson “Bioconjugate Techniques” Academic Press, California (1996),Putnam and J. Kopecek “Polymer Conjugates with Anticancer Activity”Advances in Polymer Science (1995) 122, 55-123. Specific examplesthereof can include a manner in which the anti-ALK2 antibody ischemically conjugated with the therapeutic drug for ectopic ossificationeither directly or via a spacer such as an oligopeptide, and a manner inwhich the anti-ALK2 antibody is conjugated with the therapeutic drug forectopic ossification via an appropriate drug carrier. Examples of thedrug carrier can include liposomes and water-soluble polymers. Examplesof such a manner via the drug carrier can more specifically include amanner in which the therapeutic drug for ectopic ossification isencapsulated in a liposome and the liposome is conjugated with theantibody, and a manner in which the therapeutic drug for ectopicossification is chemically conjugated with a water-soluble polymer(compound having a molecular weight on the order of 1000 to 100,000)either directly or via a spacer such as an oligopeptide and thewater-soluble polymer is conjugated with the antibody. The conjugationof the antibody (or the fragment) with the therapeutic drug for ectopicossification or the drug carrier (e.g., a liposome or a water-solublepolymer) can be carried out by a method well known to those skilled inthe art, such as a method described in G. T. Hermanson “BioconjugateTechniques” Academic Press, California (1996), and Putnam and J. Kopecek“Polymer Conjugates with Anticancer Activity” Advances in PolymerScience (1995) 122, 55-123. The encapsulation of the therapeutic drugfor ectopic ossification in the liposome can be carried out by a methodwell known to those skilled in the art, such as a method described in,for example, D. D. Lasic “Liposomes: From Physics to Applications”,Elsevier Science Publishers B.V., Amsterdam (1993). The conjugation ofthe therapeutic drug for ectopic ossification with the water-solublepolymer can be carried out by a method well known to those skilled inthe art, such as a method described in D. Putnam and J Kopecek “PolymerConjugates with Anticancer Activity” Advances in Polymer Science (1995)122, 55-123. The conjugate of the antibody (or the fragment) with theprotein as a therapeutic drug for ectopic ossification (or a fragmentthereof) can be prepared by any of the methods described above or agenetic engineering method well known to those skilled in the art.

The present invention also provides a pharmaceutical compositioncomprising a therapeutically and/or prophylactically effective amount ofthe anti-ALK2 antibody and a pharmaceutically acceptable diluent,carrier, solubilizer, emulsifier, preservative, and/or auxiliary agent.

The present invention also provides a pharmaceutical compositioncomprising a therapeutically and/or prophylactically effective amount ofthe anti-ALK2 antibody, a therapeutically and/or prophylacticallyeffective amount of at least one therapeutic drug for ectopicossification, and a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative, and/or auxiliary agent.

It is preferred that the pharmaceutically acceptable material used inthe pharmaceutical composition of the present invention should benontoxic to a recipient of the pharmaceutical composition, preferably interms of a dose or an administered concentration.

The pharmaceutical composition of the present invention can comprisepharmaceutical materials for changing or maintaining pH, osmoticpressure, viscosity, transparency, color, tonicity, sterility,stability, solubility, sustained release, absorbability, orpermeability. Examples of the pharmaceutical materials can include, butare not limited to, the following: amino acids such as glycine, alanine,glutamine, asparagine, arginine, and lysine; antimicrobial agents;antioxidants such as ascorbic acid, sodium sulfate, and sodiumbisulfite; buffers such as phosphate, citrate, or borate buffers, sodiumbicarbonate, and Tris-HCl solutions; fillers such as mannitol andglycine; chelating agents such as ethylenediaminetetraacetic acid(EDTA); complexing agents such as caffeine, polyvinylpyrrolidine,β-cyclodextrin, and hydroxypropyl-β-cyclodextrin; bulking agents such asglucose, mannose, and dextrin; other carbohydrates such asmonosaccharides and disaccharides; coloring agents; corrigents;diluents; emulsifiers; hydrophilic polymers such aspolyvinylpyrrolidine; low-molecular-weight polypeptides; salt-formingcounterions; antiseptics such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, and hydrogen peroxide;solvents such as glycerin, propylene glycol, and polyethylene glycol;sugar alcohols such as mannitol and sorbitol; suspending agents;surfactants such as sorbitan ester, polysorbates such as polysorbate 20and polysorbate 80, triton, tromethamine, lecithin, and cholesterol;stability enhancers such as sucrose and sorbitol; elasticity enhancerssuch as sodium chloride, potassium chloride, mannitol, and sorbitol;transport agents; excipients; and/or pharmaceutical auxiliary agents.The amount of these pharmaceutical materials added is preferably 0.01 to100 times, particularly, 0.1 to 10 times relative to the weight of theanti-ALK2 antibody. The suitable components of the pharmaceuticalcomposition in a preparation can be appropriately determined by thoseskilled in the art according to an applicable disease, an applicableadministration route, etc.

The excipient or the carrier in the pharmaceutical composition may beliquid or solid. Appropriate excipients or carriers may be injectablewater, physiological saline, artificial cerebrospinal fluids, or othermaterials usually used in parenteral administration. Neutralphysiological saline or physiological saline containing serum albuminmay be used as a carrier. The pharmaceutical composition can contain aTris buffer of pH 7.0 to 8.5, an acetate buffer of pH 4.0 to 5.5, or acitrate buffer of pH 3.0 to 6.2. These buffers can also contain sorbitolor other compounds. Examples of the pharmaceutical composition of thepresent invention can include a pharmaceutical composition comprisingthe anti-ALK2 antibody, and a pharmaceutical composition comprising theanti-ALK2 antibody and at least one therapeutic drug for ectopicossification. The pharmaceutical composition of the present invention isprepared in the form of a freeze-dried product or a liquid as a drughaving a selected recipe and a necessary purity. The pharmaceuticalcomposition comprising the anti-ALK2 antibody, or the pharmaceuticalcomposition comprising the anti-ALK2 antibody and at least onetherapeutic drug for ectopic ossification can also be formed as afreeze-dried product using an appropriate excipient such as sucrose.

The pharmaceutical composition of the present invention may be preparedfor parenteral administration or may be prepared for absorption in thegastrointestinal tract through an oral route. The recipe andconcentration of the preparation can be determined according to anadministration method. As the anti-ALK2 antibody contained in thepharmaceutical composition of the present invention has higher affinityfor ALK2, i.e., a lower dissociation constant (KD value) for ALK2, theanti-ALK2 antibody can exert efficacy even at a reduced dose to a human.Therefore, the dose of the pharmaceutical composition of the presentinvention to a human can also be determined on the basis of this result.The dose for the administration of the human type anti-ALK2 antibody toa human is, for example, approximately 0.1 to 100 mg/kg, which can beadministered once or twice or more per 1 to 180 days. However, the doseand the number of doses should generally be determined in considerationof the sex, body weight, and age of a patient, symptoms, severity,adverse reactions, etc., and therefore, are not limited to the dose orusage described above.

Non-limiting examples of the form of the pharmaceutical composition ofthe present invention can include injections including intravenousdrips, suppositories, transnasal formulations, sublingual formulations,and transdermal absorption formulations. The administration route is anoral administration route or a parenteral administration route.Non-limiting examples of the parenteral administration route includeintravenous, intraarterial, intramuscular, intrarectal, transmucosal,and intradermal routes.

EXAMPLES

The present invention will be specifically described hereinafter withExamples; however, the invention is not limited thereto. In thefollowing Examples, unless otherwise specified, operations concerninggenetic manipulation were performed in accordance with methods describedin “Molecular Cloning” (Sambrook, J., Fritsch, E. F., and Maniatis, T.,Cold Spring Harbor Laboratory Press, 1989), or where commercial reagentsor kits were used, they were used in accordance with the manuals forsuch commercial products.

Example 1

Preparation of Rat Anti-ALK2 Antibodies (11E2, 15A6, 25C11, and 27D11:Hereinafter Abbreviated as A2-11E, A2-15A, A2-25C, and A2-27D)

1)-1 Preparation of an Antigen

Mouse ALK2-His&Fc (Cat. #50297-M03H) from Sino Biological Inc. was usedas mALK2-Fc, which is the antigen.

1)-2 Immunization of Animals

1 mg/mL of mouse ALK2-His&Fc was mixed with an equal volume ofTiterMaxGold (TiterMax, USA) to prepare an emulsion. To two 6-week-oldWister female rats, 100 μg per rat of the antigen was subcutaneouslyadministered with the adjuvant in two divided doses. After 1 to 2 weeks,40 μg of only the antigen solution was subcutaneously administered, andafter 3 days, spleen cells and various lymph glands were asepticallyremoved as antibody-producing cells, and subjected to the following cellfusion.

1)-3 Cell Fusion with Myeloma Cells

The above-described antibody-producing cells were mixed with a myelomacell line

(P3U1 cells) derived from BALB/c mice at a ratio of 5:1 to 10:1, andfused for 3 minutes using 50% polyethylene glycol 1500, and then dilutedover 6 minutes. The fused cells were seeded with feeder cells(thymocytes) into ten 96-well plates per rat. The cells were cultured in15% FBS-containing RPMI1640 medium (containing glutamine, pyruvic acid,penicillin, and streptomycin) containing HAT supplement.

1)-4 Selection of Hybridomas

One week after the cell fusion, using each culture supernatant, clonesrecognizing mouse ALK2-His&Fc used as the antigen were selected by theenzyme-linked immunosorbent assay (ELISA), and clones recognizing onlyhuman Fc (Sino Biological Inc.) were excluded. ELISA was performed asfollows.

First, the antigen diluted to 1 μg/ml with PBS was immobilized onto a96-well ELISA plate (NUNC, Cat. #442404) for 2 hours at room temperatureor overnight at 4° C. The immobilized solution was removed, and theplate was blocked with a 0.5% skim milk solution dissolved in PBS for 30minutes at room temperature. Next, the above-described culturesupernatant of hybridomas was added, and the plate was left for 1 hourat room temperature. After the plate was washed, an ALP-labeled anti-ratIgG antibody (SBA, Inc.) diluted with 0.5% skim milk to 1:2500 wasadded, and the plate was further left for 1 hour at room temperature.After the plate was washed, a phenyl phosphate substrate was reacted for20 minutes at room temperature, and then absorbance at a wavelength of492 nm was measured.

Selected hybridomas were cloned twice or more by limiting dilution.Through the above-described operations, hybridoma cell lines producingmonoclonal antibodies A2-11E, A2-15A, A2-25C, and A2-27D were isolated.

The results are shown in FIG. 1. The results showed that each of themonoclonal antibodies produced by hybridomas A2-11E, A2-15A, A2-25C, andA2-27D recognized mouse ALK2-His&Fc, and did not bind to human Fc.

Example 2

In vitro evaluation of rat anti-ALK2 antibodies (A2-11E, A2-15A, A2-25C,and A2-27D)

2)-1 Antibody Screening by Flow Cytometry

2)-1-1 Preparation of Mouse and Human ALK2-Expressing Cells

HEK293A cells were seeded into a 100-mm dish at 3×10⁴ cells/cm², andcultured overnight in 15% FBS-containing DMEM medium under theconditions of 5% CO₂ at 37° C. On the following day, the HEK293A cellswere transfected with each of pcDEF3/mouse ALK2(WT)-EGFP, pcDEF3/humanALK2(WT)-EGFP, pcDEF3/human ALK2 (R206H)-EGFP, and pcDEF3, usingLipofectamine 2000 (Invitrogen), and further cultured overnight. On thefollowing day, 100 μL each of the cell suspensions adjusted to 1×10⁶cells/mL was dispensed into a 1.5-mL microfuge and centrifuged at 500 gfor 5 minutes, and then the supernatant was removed. To the cells, 100μL of purified IgG diluted to 1 μg/mL was added, and the cells were leftfor 30 minutes at 4° C. After the cells were washed with PBS threetimes, 100 μL of Alexa flour 647 Goat anti-Rat IgG (H+L) (LifeTechnologies) diluted to 1:200 was added, and the cells were furtherleft for 30 minutes at 4° C. After the cells were washed with PBS threetimes, fluorescence was detected by a flow cytometer (FACS Aria II: BDBiosciences). The data was analyzed using BD Diva Software (BDBiosciences), and the intensity of EGFP expressed by the cells and thefluorescence intensity of stained Alexa flour 647 were plotted.

The results are shown in FIGS. 2A to 2D. It was confirmed that each ofthe monoclonal antibodies produced by hybridomas A2-11E, A2-15A, A2-25C,and A2-27D did not recognize fluorescence protein EGFP-expressing cells(FIG. 2A), but specifically recognized mouse ALK2-expressing cells (FIG.2B) as well as wild-type and FOP human ALK2-expressing cells (FIGS. 2Cand 2D, respectively). These results show that the monoclonal antibodiesproduced by hybridomas A2-11E, A2-15A, A2-25C, and A2-27D are antibodiesthat bind to the extracellular region of ALK2.

2)-2 Antibody Screening by Immunostaining

2)-2-1 Preparation of Human ALK1-, ALK2-, ALK3-, and ALK6-ExpressingCells

Mouse C2C12 cells were seeded into a 96-well plate (Greiner Bio-One) at5×10³ cells/well, and cultured overnight in 15% FBS-containing DMEMmedium under the conditions of 5% CO₂ at 37° C. On the following day,the C2C12 cells were transfected with each of pcDEF3/human ALK1-EGFP,pcDEF3/human ALK2-EGFP, pcDEF3/human ALK3-EGFP, pcDEF3/human ALK6-EGFP,and pcDEF3 as a control, using Lipofectamine 2000 (Invitrogen), andfurther cultured overnight.

2)-2-2 Immunofluorescence Staining of Human ALK, ALK2, ALK3, and ALK6

The C2C12 cells were fixed with 10% neutral formalin (Nacalai Tesque,Japan) for 20 minutes at room temperature, and then blocked with 10%goat normal serum for 30 minutes. After the blocking agent was removed,purified IgG diluted to 10 μg/mL with the blocking agent was added, andthe cells were left for 1 hour at room temperature. After the cells werewashed with PBS three times, Goat Alexa 594-conjugated anti-rat IgG(Invitrogen) diluted to 1:1000 with the blocking agent was added, andthe cells were left for 1 hour at room temperature. After the cells werewashed with PBS three times, the cells were fixed with 10% neutralformalin for 15 minutes at room temperature, and nuclear-stained withDAPI (LifeTechnologies). Fluorescent signals were analyzed with afluorescence microscope BZ-9000 (Keyence).

The results are shown in FIG. 3. It was confirmed that the monoclonalantibodies produced by hybridomas A2-11E, A2-15A, A2-25C, and A2-27Dspecifically recognized only ALK2-expressing cells, and did notrecognize ALK1-, ALK3-, and ALK6-expressing cells. These results showthat the monoclonal antibodies produced by hybridomas A2-11E, A2-15A,A2-25C, and A2-27D are antibodies that specifically bind to ALK2.

2)-2-3 Preparation of Cells Expressing ALK2 Mutants Identified in FOP

Mouse C2C12 cells were seeded into a 96-well plate (Greiner Bio-One) at0.5×10³ cells/well, and cultured in 15% FBS-containing DMEM mediumovernight under the conditions of 5% CO₂ at 37° C. On the following day,the C2C12 cells were transfected with each of a wild-type human ALK2cDNA integrated into pcDEF3; pcDEF3/human ALK2-L 196P, pcDEF3/humanALK2-P 197_F198delinsL, pcDEF3/human ALK2-R202I, pcDEF3/humanALK2-R206H, pcDEF3/human ALK2-Q207E, pcDEF3/human ALK2-R258S,pcDEF3/human ALK2-G325A, pcDEF3/human ALK2-G328E, pcDEF3/humanALK2-G328R, pcDEF3/human ALK2-G328W, pcDEF3/human ALK2-G356D, andpcDEF3/human ALK2-R375P, which are variants identified in FOP; andpcDEF3 as a control, using Lipofectamine 2000 (Invitrogen), and furthercultured overnight.

2)-2-4 Immunofluorescence Staining of ALK2 Mutants Identified in FOP

The C2C12 cells were fixed with 10% neutral formalin (Nacalai Tesque,Japan) for 20 minutes at room temperature, and then blocked with 10%goat normal serum for 30 minutes. After the blocking agent was removed,purified IgG diluted to 10 μg/mL with the blocking agent was added, andthe cells were left for 1 hour at room temperature. After the cells werewashed with PBS three times, Goat Alexa 594-conjugated anti-rat IgG(Invitrogen) diluted to 1:1000 with the blocking agent was added, andthe cells were left for 1 hour at room temperature. After the cells werewashed with PBS three times, the cells were fixed with 10% neutralformalin for 15 minutes at room temperature, and nuclear-stained withDAPI (LifeTechnologies). Fluorescent signals were analyzed with afluorescence microscope BZ-9000 (Keyence).

The results are shown in FIG. 4. It was confirmed that the monoclonalantibodies produced by hybridomas A2-11E, A2-15A, A2-25C, and A2-27Drecognized the cells expressing the 12 types of ALK2 mutants identifiedin FOP, in addition to the wild-type ALK2-expressing cells. Theseresults show that the monoclonal antibodies produced by hybridomasA2-11E, A2-15A, A2-25C, and A2-27D are antibodies that bind to wild-typeALK2 and each of the ALK2 mutants in FOP.

2)-3 Antibody Screening by Luciferase Reporter Assay

The ALK2-mediated intracellular signaling inhibitory activity of each ofthe monoclonal antibodies was analyzed using a BMP-specific luciferasereporter. HEK293A cells were seeded into a 96-well white plate forluciferase assay (Greiner Bio-One) at 1×10⁴ cells/well, and culturedovernight in 15% FBS-containing DMEM medium under the conditions of 5%CO₂ at 37° C. On the following day, the HEK293A cells were transfectedwith pcDEF3/human ALK2(WT)-V5-His or pcDEF3/human ALK2(R206H)-V5-His,pGL4.26/Id1WT4F-luc (Genes Cells, 7, 949 (2002)), and phRL SV40(Promega), using Lipofectamine 2000 (Invitrogen). After 2.5 hours, themedium was replaced with fresh OPTI-MEM I (LifeTechnologies), and thecells were further cultured for 1 hour. Thereafter, the medium wasreplaced with OPTI-MEM I containing the serially diluted monoclonalantibody and 10 ng/mL of BMP7 (Milteney) or 0.5 ng/mL of BMP9(Peprotech), and the cells were further cultured overnight. On thefollowing day, the firefly and Renilla luciferase activities weremeasured with the plate reader GENios (TECAN), using Dual-Glo LuciferaseAssay System (Promega).

The results are shown in FIG. 5. It was confirmed that the monoclonalantibodies produced by hybridomas A2-11E, A2-15A, A2-25C, and A2-27Dsuppressed the BMP7-induced BMP-specific luciferase activity in adose-dependent manner, in the HEK293A cells overexpressing wild-typeALK2 and mutant R206H.

2)-4 Antibody Screening by BMP-Induced Osteoblast DifferentiationInduction Assay

The endogenous ALK2-mediated intracellular signaling inhibitory activityof each of the monoclonal antibodies was analyzed based on the effectupon the BMP-induced activity of inducing the differentiation of C2C12cells into osteoblasts. C2C12 cells were seeded into a 96-well plate(Greiner Bio-One) at 0.5×10³ cells/well, and cultured in 15%FBS-containing DMEM medium overnight under the conditions of 5% C02 at37° C. On the following day, the medium was replaced with OPTI-MEM Icontaining the serially diluted monoclonal antibody and 200 ng/mL ofBMP2 (Corefront Corporation), 200 ng/mL of BMP7 (Miteney), or 20 ng/mLof GDF2/BMP9 (Peprotech), and the cells were further cultured for 3days. After the medium was removed from the C2C12 cells and the cellswere washed with PBS, the cells were treated with 50 μL/well of anice-cold acetone:ethanol (1:1) solution for 1 minute, and further washedwith PBS three times. ALP activity was measured as an index of thedifferentiation into cells for forming osteoblasts. To measure ALPactivity, 100 μL/well of a substrate solution (0.1 M diethanolamine(Sigma-Aldrich)-HCl, pH 10.0, containing 1 mg/mL of 4-nitrophenylphosphate (Sigma-Aldrich) and 1 mM of MgCl₂) was added, and reacted for15 to 30 minutes at room temperature on an agitation shaker. Thereaction was stopped by the addition of 50 μL of 3M NaOH, and absorbanceat a wavelength of 405 nm was measured using the microplate readerInfinite F50 (TECAN).

The results are shown in FIGS. 6 and 7. It was confirmed that themonoclonal antibodies produced by hybridomas A2-11E, A2-15A, A2-25C, andA2-27D barely suppressed the BMP2-induced differentiation of the C2C12cells into osteoblast-like cells (FIG. 6), but suppressed the BMP7- andGDF2/BMP9-induced differentiation of the C2C12 cells intoosteoblast-like cells in a dose-dependent manner (FIG. 7). These resultsshow that the monoclonal antibodies produced by hybridomas A2-11E,A2-15A, A2-25C, and A2-27D are antibodies that suppress endogenous ALK2physiologically expressed by the C2C12 cells.

Example 3

Nucleotide sequencing of cDNAs encoding variable regions of ratanti-ALK2 antibodies (A2-11E, A2-15A, A2-25C, and A2-27D)

3)-1 Nucleotide Sequencing of cDNAs Encoding Variable Regions of A2-11E

3)-1-1 Preparation of Total RNA from A2-11E-Producing Hybridomas

To amplify cDNAs containing variable regions of A2-11E, total RNA wasprepared from the A2-11E-producing hybridomas, using TRIzol Reagent(Ambion).

3)-1-2 Synthesis of a cDNA (5′-RACE-Ready cDNA)

A cDNA (5′-RACE-Ready cDNA) was synthesized from 1 μg of the total RNAprepared in Example 3)-1-1, using SMARTer RACE cDNA Amplification Kit(Clontech).

3)-1-3 Amplification by 5′-RACE PCR and Sequencing of a cDNA Containinga Heavy Chain Variable Region of A2-11E

UPM (Universal Primer A Mix: included in SMARTer RACE cDNA AmplificationKit) and an oligonucleotide having the sequence:5′-CTCCAGAGTTCCAGGTCACGGTGACTGGC-3′ (RG2AR3: SEQ ID NO: 88) were used asprimers for PCR amplification of a cDNA containing the variable regionof the heavy chain gene of A2-11E. UPM included in SMARTer RACE cDNAAmplification Kit (Clontech) was used, and RG2AR3 was designed from thesequence of a rat heavy chain constant region on a database.

A cDNA containing a heavy chain variable region of A2-11E was amplifiedby 5′-RACE PCR using this combination of primers and the cDNA(5′-RACE-Ready cDNA) synthesized in Example 3)-1-2 as a template. PCRwas performed using KOD-Plus-(TOYOBO, Japan) as polymerase, and usingthe touch-down PCR program in accordance with the manual of SMARTer RACEcDNA Amplification Kit (Clontech).

The cDNA containing the heavy chain variable region amplified by 5′-RACEPCR was purified using MinElute PCR Purification Kit (QIAGEN), and thencloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and thesequence analysis of the nucleotide sequence of the cloned cDNAcontaining the heavy chain variable region was performed.

As sequence primers, the oligonucleotide having the sequence:5′-CTCCAGAGTTCCAGGTCACGGTGACTGGC-3′ (RG2AR3: SEQ ID NO: 88) designedfrom the sequence of a rat heavy chain constant region on a database andNUP (Nested Universal Primer A: included in SMARTer RACE cDNAAmplification Kit) were used.

The sequence analysis was performed using a gene sequence analyzer (“ABIPRISM 3700 DNA Analyzer; Applied Biosystems” or “Applied Biosystems3730xl Analyzer; Applied Biosystems”), and the sequence reaction wasperformed using GeneAmp 9700 (Applied Biosystems).

The determined nucleotide sequence of the cDNA encoding the heavy chainvariable region of A2-11E is shown in SEQ ID NO: 1 in the SequenceListing, and the amino acid sequence is shown in SEQ ID NO: 2.

3)-1-4 Amplification by 5′-RACE PCR and Sequencing of a cDNA Containinga Light Chain Variable Region of A2-11E

UPM (Universal Primer A Mix: included in SMARTer RACE cDNA AmplificationKit) and an oligonucleotide having the sequence:5′-TCAGTAACACTGTCCAGGACACCATCTC-3′ (RKR5; SEQ ID NO: 89) were used asprimers for PCR amplification of a cDNA containing the variable regionof the light chain gene of A2-11E. UPM included in SMARTer RACE cDNAAmplification Kit (Clontech) was used, and RKR5 was designed from thesequence of a rat light chain constant region on a database.

A cDNA containing a light chain variable region of A2-11E was amplifiedby 5′-RACE PCR using this combination of primers and the cDNA(5′-RACE-Ready cDNA) synthesized in Example 3)-1-2 as a template. PCRwas performed using KOD-Plus- (TOYOBO, Japan) as polymerase, and usingthe touch-down PCR program in accordance with the manual of SMARTer RACEcDNA Amplification Kit (Clontech).

The cDNA containing the light chain variable region amplified by 5′-RACEPCR was purified using MinElute PCR Purification Kit (QIAGEN), and thencloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and thesequence analysis of the nucleotide sequence of the cloned cDNAcontaining the light chain variable region was performed.

As a sequence primer, the following sequence was designed from thesequence of a rat light chain constant region on a database. Theoligonucleotide having the designed sequence:5′-TCAGTAACACTGTCCAGGACACCATCTC-3′ (RKR5; SEQ ID NO: 89) and NUP (NestedUniversal Primer A: included in SMARTer RACE cDNA Amplification Kit)were used.

The sequence analysis was performed using a gene sequence analyzer (“ABIPRISM 3700 DNA Analyzer; Applied Biosystems” or “Applied Biosystems3730xl Analyzer; Applied Biosystems”), and the sequence reaction wasperformed using GeneAmp 9700 (Applied Biosystems).

The determined nucleotide sequence of the cDNA encoding the light chainvariable region of A2-11E is shown in SEQ ID NO: 3 in the SequenceListing, and the amino acid sequence is shown in SEQ ID NO: 4.

3)-2 Nucleotide Sequencing of cDNAs Encoding Variable Regions of A2-15A

3)-2-1 Preparation of Total RNA from A2-15A-Producing Hybridomas

To amplify cDNAs containing variable regions of A2-15A, total RNA wasprepared from the A2-15A-producing hybridomas, as in Example 3)-1-1.

3)-2-2 Synthesis of a cDNA (5′-RACE-Ready cDNA)

A cDNA (5′-RACE-Ready cDNA) was synthesized from 1 μg of the total RNAprepared in Example 3)-2-1, as in Example 3)-1-2.

3)-2-3 Amplification by 5′-RACE PCR and Sequencing of a cDNA Containinga Heavy Chain Variable Region of A2-15A

A cDNA containing a heavy chain variable region of A2-15A was amplifiedas in Example 3)-1-3, using the cDNA (5′-RACE-Ready cDNA) synthesized inExample 3)-2-2 as a template, and the sequence was determined.

The determined nucleotide sequence of the cDNA encoding the heavy chainvariable region of A2-15A is shown in SEQ ID NO: 5 in the SequenceListing, and the amino acid sequence is shown in SEQ ID NO: 6.

3)-2-4 Amplification by 5′-RACE PCR and Sequencing of a cDNA Containinga Light Chain Variable Region of A2-15A

A cDNA containing a light chain variable region of A2-15A was amplifiedas in Example 3)-1-4, using the cDNA (5′-RACE-Ready cDNA) synthesized inExample 3)-2-2 as a template, and the sequence was determined.

The determined nucleotide sequence of the cDNA encoding the light chainvariable region of A2-15A is shown in SEQ ID NO: 7 in the SequenceListing, and the amino acid sequence is shown in SEQ ID NO: 8.

3)-3 Nucleotide Sequencing of cDNAs Encoding Variable Regions of A2-25C

3)-3-1 Preparation of Total RNA from A2-25C-Producing Hybridomas

To amplify cDNAs containing variable regions of A2-25C, total RNA wasprepared from the A2-25C-producing hybridomas, as in Example 3)-1-1.

3)-3-2 Synthesis of a cDNA (5′-RACE-Ready cDNA)

A cDNA (5′-RACE-Ready cDNA) was synthesized from 1 gig of the total RNAprepared in Example 3)-3-1, as in Example 3)-1-2.

3)-3-3 Amplification by 5′-RACE PCR and Sequencing of a cDNA Containinga Heavy Chain Variable Region of A2-25C

A cDNA containing a heavy chain variable region of A2-25C was amplifiedas in Example 3)-1-3, using the cDNA (5′-RACE-Ready cDNA) synthesized inExample 3)-3-2 as a template, and the sequence was determined.

The determined nucleotide sequence of the cDNA encoding the heavy chainvariable region of A2-25C is shown in SEQ ID NO: 9 in the SequenceListing, and the amino acid sequence is shown in SEQ ID NO: 10.

3)-3-4 Amplification by 5′-RACE PCR and Sequencing of a cDNA Containinga Light Chain Variable Region of A2-25C

A cDNA containing a light chain variable region of A2-25C was amplifiedas in Example 3)-1-4, using the cDNA (5′-RACE-Ready cDNA) synthesized inExample 3)-3-2 as a template, and the sequence was determined.

The determined nucleotide sequence of the cDNA encoding the light chainvariable region of A2-25C is shown in SEQ ID NO: 11 in the SequenceListing, and the amino acid sequence is shown in SEQ ID NO: 12.

3)-4 Nucleotide Sequencing of cDNAs Encoding Variable Regions of A2-27D

3)-4-1 Preparation of Total RNA from A2-27D-Producing Hybridomas

To amplify cDNAs containing variable regions of A2-27D, total RNA wasprepared from the A2-27D-producing hybridomas, as in Example 3)-1-1.

3)-4-2 Synthesis of a cDNA (5′-RACE-Ready cDNA)

A cDNA (5′-RACE-Ready cDNA) was synthesized from 1 μg of the total RNAprepared in Example 3)-4-1, as in Example 3)-1-2.

3)-4-3 Amplification by 5′-RACE PCR and Sequencing of a cDNA Containinga Heavy Chain Variable Region of A2-27D

A cDNA containing a heavy chain variable region of A2-27D was amplifiedas in Example 3)-1-3, using the cDNA (5′-RACE-Ready cDNA) synthesized inExample 3)-4-2 as a template, and the sequence was determined.

The determined nucleotide sequence of the cDNA encoding the heavy chainvariable region of A2-27D is shown in SEQ ID NO: 13 in the SequenceListing, and the amino acid sequence is shown in SEQ ID NO: 14.

3)-4-4 Amplification by 5′-RACE PCR and Sequencing of a cDNA Containinga Light Chain Variable Region of A2-27D

A cDNA containing a light chain variable region of A2-27D was amplifiedas in Example 3)-1-4, using the cDNA (5′-RACE-Ready cDNA) synthesized inExample 3)-4-2 as a template, and the sequence was determined.

The determined nucleotide sequence of the cDNA encoding the light chainvariable region of A2-27D is shown in SEQ ID NO: 15 in the SequenceListing, and the amino acid sequence is shown in SEQ ID NO: 16.

Example 4

In vivo evaluation of rat anti-ALK2 antibodies (A2-15A and A2-27D)

4)-1 Preparation of Hybridoma Culture Supernatants

1.0×10⁶ hybridomas obtained in Example 1)-4 were cultured in 10%FBS-containing TIL high-glucose medium (T75 flask), and then cultured athigh density using INTEGRA CL1000 (10% FBS medium). Next, the medium wasreplaced with a serum-free medium, the cells were further cultured inINTEGRA CL1000, and then required volumes of hybridoma culturesupernatants were obtained. The obtained hybridoma culture supernatantswere stored at 2 to 8° C. until they were subjected to purification.

4)-2 Purification of the Antibodies from the Hybridoma CultureSupernatants

The antibodies were purified from the culture supernatants obtained inExample 4)-1 by protein G affinity chromatography (at 4 to 6° C.) in onestep. The buffer replacement step after the purification by protein Gaffinity chromatography was performed at 4 to 6° C. Initially, eachculture supernatant of hybridomas was applied to a PBS-equilibratedcolumn packed with protein G (GE Healthcare Bioscience). After the entryof the whole culture supernatant in the column, the column was washedwith at least twice the column volume of PBS. Next, fractions containingthe antibody were collected by elution with a 0.1 M aqueousglycine/hydrochloric acid solution (pH 2.7). The collected fractionswere adjusted to pH 7.0 to 7.5 by the addition of 1 M Tris-HCl (pH 9.0),and then subjected to buffer replacement with HBSor (25 mM histidine/5%sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer DialysisCassette). The fractions were concentrated using Centrifugal UF FilterDevice VIVASPIN20 (molecular weight cutoff: UF10K, Sartorius, at 4° C.),and adjusted to an IgG concentration of 8 mg/ml or more. Finally, thefractions were filtered using the Minisart-Plus filter (Sartorius) toobtain a purified sample.

4)-3 BMP Transplantation-Induced Heterotopic Ossification InductionModels

The heterotopic ossification-suppressing activity of the monoclonalantibodies in skeletal muscle tissue was analyzed by BMP-inducedheterotopic ossification induction experiments in mice. BMP-containingpellets were prepared by impregnating a type I collagen sponge(Collatape, Zimmer Dental) adjusted to a diameter of 4 mm with 1 gig ofBMP7 (Milteney) or 1 μg of GDF2/BMP9 (Peprotech), and freeze-drying thesponge overnight in a freeze-drying machine (FDU-810, Tokyo RikakikaiCo., Ltd., Japan). 8- to 10-week-old C57BL/6 mice were placed undergeneral anesthesia, using a simple inhalation anesthesia apparatus forsmall animal experiments (Natsume Seisakusho Co., Ltd., Japan) and 2%isoflurane (Wako Pure Chemical Industries, Ltd., Japan), and one of theBMP-containing pellets was transplanted into the thigh muscle tissue ofeach of the left and right lower extremities. The mice receivedsubcutaneous administration of the monoclonal antibodies produced byhybridomas A2-15A and A2-27D at 10 mg/kg once a week, from 1 week beforethe transplantation until week 2 after the transplantation, as they wereraised. An equal volume of a solvent (25 mM Histidine/5% Sorbitol, pH6.0) was administered to a control group. Two weeks after thetransplantation of the BMP-containing pellets, the thigh bone with thetransplanted BMP-containing pellets were extracted, and heterotopicossification was analyzed by micro-CT (μCT35, SCANCO).

The results are shown in FIG. 8. The micro-CT analysis showed thatheterotopic ossification was induced on the right-hand side of the thighbone in the mice transplanted with BMP7 and GDF2/BMP9 and treated withthe vehicle. In contrast, heterotopic ossification was not detected inthe mice that received the administration of the monoclonal antibodiesproduced by hybridomas A2-15A and A2-27D, although they weretransplanted with BMP7 and GDF2/BMP9. It was thus confirmed that themonoclonal antibodies produced by hybridomas A2-15A and A2-27Dsuppressed the induction of heterotopic ossification in the skeletalmuscle tissue induced by BMP7 and GDF2/BMP9.

Example 5

Preparation of Human Chimeric Anti-ALK2 Antibodies (cA2-15A and cA2-27D)

5)-1 Construction of a Chimeric and Humanized Antibody Light ChainExpression Vector pCMA-LK

A fragment of about 5.4 kb obtained by digesting the plasmidpcDNA3.3-TOPO/LacZ (Invitrogen) with restriction enzymes XbaI and PmeIand a DNA fragment containing a DNA sequence encoding a human κ chainsecretion signal and a human κ chain constant region shown in the SEQ IDNO: 17 were ligated using In-Fusion Advantage PCR Cloning Kit (Clontech)to prepare pcDNA3.3/LK.

pcDNA3.3/LK was used as a template in PCR using the following primerset. The obtained fragment of about 3.8 kb was phosphorylated and thenself-ligated, thereby constructing a chimeric and humanized antibodylight chain expression vector pCMA-LK having a signal sequence, acloning site, and a human κ chain constant region downstream of the CMVpromoter.

Primer set: (3.3-F1; SEQ ID NO: 90) 5′-TATACCGTCGACCTCTAGCTAGAGCTTGGC-3′(3.3-R1; SEQ ID NO: 91) 5′-GCTATGGCAGGGCCTGCCGCCCCGACGTTG-3′

5)-2 Construction of a Chimeric and Humanized Antibody IgG1 Type HeavyChain Expression Vector pCMA-G1

A DNA fragment lacking the human κ chain secretion signal and the humanκ chain constant region by digesting pCMA-LK with XbaI and PmeI and aDNA fragment containing a DNA sequence encoding amino acids of a humanheavy chain signal sequence and a human IgG1 constant region of SEQ IDNO: 18 were ligated using In-Fusion Advantage PCR Cloning Kit(Clontech), thereby constructing a chimeric and humanized antibody IgG1type heavy chain expression vector pCMA-G1 having a signal sequence, acloning site, and a human IgG1 heavy chain constant region downstream ofthe CMV promoter.

5)-3 Construction of a cA2-15A Heavy Chain Expression Vector

Using the cDNA containing the heavy chain variable region of A2-15Aobtained in Example 3)-2-3 as a template, a DNA fragment containing thecDNA encoding the heavy chain variable region was amplified, usingKOD-Plus- (TOYOBO, Japan) and the following primer set, and insertedinto a restriction enzyme BlpI-cleaved site of the chimeric andhumanized IgG1 type heavy chain expression vector pCMA-G1, usingIn-Fusion HD PCR Cloning Kit (Clontech), thereby constructing a cA2-15Aheavy chain expression vector. The obtained expression vector wasdesignated as “pCMA-G1/cA2-15A”. The nucleotide sequence of the cA2-15Aheavy chain is shown in SEQ ID NO: 19, and the amino acid sequence isshown in SEQ ID NO: 20.

Primer set for the cA2-15A heavy chain: (A2-15AH-F; SEQ ID NO: 92)5′-CCAGATGGGTGCTGAGCGAGGTGCAGCTGGTGGAGTCTGGCGGA G-3′(A2-15AH-R; SEQ ID NO: 93)5′-CTTGGTGGAGGCTGAGCTGACAGTGACCAGAGTGCCTTGGCCCC AG-3′

5)-4 Construction of a cA2-15A Light Chain Expression Vector

Using the cDNA containing the light chain variable region of A2-15Aobtained in Example 3)-2-4 as a template, a DNA fragment containing thecDNA encoding the light chain variable region was amplified, usingKOD-Plus- (TOYOBO, Japan) and the following primer set, and insertedinto a restriction enzyme BsiWI-cleaved site of the chimeric andhumanized light chain expression general-purpose vector pCMA-LK, usingIn-Fusion HD PCR Cloning Kit (Clontech), thereby constructing a cA2-15Alight chain expression vector. The obtained expression vector wasdesignated as “pCMA-LK/cA2-15A”. The nucleotide sequence of the cA2-15Alight chain is shown in SEQ ID NO: 21 in the Sequence Listing, and theamino acid sequence is shown in SEQ ID NO: 22.

Primer set for the cA2-15A light chain: (A2-15AL-F; SEQ ID NO: 94)5′-ATCTCCGGCGCGTACGGCGACATTGTCTTGACCCAGTCTCCTGC-3′(A2-15AL-R; SEQ ID NO: 95)5′-GGAGGGGGCGGCCACAGCCCGTTTCAGTTCCAGCTTGGTCCCAG-3′

5)-5 Construction of a cA2-27D Heavy Chain Expression Vector

Using the cDNA containing the heavy chain variable region of A2-27Dobtained in Example 3)-4-3 as a template, a DNA fragment containing thecDNA encoding the heavy chain variable region was amplified usingKOD-Plus- (TOYOBO, Japan) and the following primer set, and insertedinto a restriction enzyme BlpI-cleaved site of the chimeric andhumanized IgG1 type heavy chain expression vector pCMA-G1, usingIn-Fusion HD PCR Cloning Kit (Clontech), thereby constructing a cA2-27Dheavy chain expression vector. The obtained expression vector wasdesignated as “pCMA-G1/cA2-27D”. The nucleotide sequence of the cA2-27Dheavy chain is shown in SEQ ID NO: 23 in the Sequence Listing, and theamino acid sequence is shown in SEQ ID NO: 24.

Primer set for the cA2-27D heavy chain: (A2-27DH-F; SEQ ID NO: 96)5′-CCAGATGGGTGCTGAGCGAGGTGCAGCTGGTGGAGTCTGGAGGA G-3′(A2-27DH-R; SEQ ID NO: 97)5′-CTTGGTGGAGGCTGAGCTCACGGTGACCACGGTTCCTGGGCCCC AG-3′

5)-6 Construction of a cA2-27D Light Chain Expression Vector

Using the cDNA containing the light chain variable region of A2-27Dobtained in Example 3)-4-4 as a template, a DNA fragment containing thecDNA encoding the light chain variable region was amplified usingKOD-Plus- (TOYOBO, Japan) and the following primer set, and insertedinto a restriction enzyme BsiWI-cleaved site of the chimeric andhumanized antibody light chain expression general-purpose vectorpCMA-LK, using In-Fusion HD PCR Cloning Kit (Clontech), therebyconstructing a cA2-27D antibody light chain expression vector. Theobtained expression vector was designated as “pCMA-LK/cA2-27D”. Thenucleotide sequence of the cA2-27D antibody light chain is shown in SEQID NO: 25, and the amino acid sequence is shown in SEQ ID NO: 26.

Primer set for the cA2-27D light chain: (A2-27DL-F; SEQ ID NO: 98)5′-ATCTCCGGCGCGTACGGCGAAATTGTTCTCACTCAGTCTCCAAC-3′(A2-15AL-R; SEQ ID NO: 95)5′-GGAGGGGGCGGCCACAGCCCGTTTCAGTTCCAGCTTGGTCCCAG-3′

5)-7 Production of cA2-15A and cA2-27D

FreeStyle 293F cells (Invitrogen) were subcultured and cultured inaccordance with the manual. 1.2×10⁹ FreeStyle 293F cells (Invitrogen) inthe logarithmic growth phase were seeded into 3 L Fernbach ErlenmeyerFlask (Corning), and diluted with the FreeStyle293 expression medium(Invitrogen) to 2.0×10⁶ cells/ml, and then the cells were cultured withshaking at 90 rpm for 1 hour in an 8% C02 incubator at 37° C. 1.8 mg ofpolyethyleneimine (Polyscience #24765) was dissolved in 20 ml of theOpti-Pro SFM medium (Invitrogen). Then, the H chain expression vector(0.24 mg) and the L chain expression vector (0.36 mg) prepared usingNucleoBond Xtra (TaKaRa, Japan) were added into 20 ml of the Opti-ProSFM medium (Invitrogen). 20 ml of the expression vector/Opti-Pro SFMmixed solution was added into 20 ml of the polyethyleneimine/Opti-ProSFM mixed solution, and the mixture was gently stirred, further left for5 minutes, and then added into the FreeStyle 293F cells. A culturesupernatant obtained by shake culture at 90 rpm for 4 hours in an 8% C02incubator at 37° C., followed by the addition of 600 ml of the EX-CELLVPRO medium (SAFC Biosciences), 18 ml of GlutaMAX I (GIBCO), and 30 mlof Yeastolate Ultrafiltrate (GIBCO), and by shake culture at 90 rmp for7 days in an 8% C02 incubator at 37° C., was filtered through DisposableCapsule Filter (Advantec #CCS-045-E1H).

The chimeric antibody of the rat antibody A2-15A obtained by thecombination of pCMA-G1/cA2-15A and pCMA-LK/cA2-15A was designated as“cA2-15A”, and the chimeric antibody of the rat antibody A2-27D obtainedby the combination of pCMA-G1/cA2-27D and pCMA-LK/cA2-27D was designatedas “cA2-27D”.

5)-8 Two-Step Purification of cA2-15A and cA2-27D

Each of the antibodies was purified from the culture supernatantobtained in Example 5)-7 by two steps using rProtein A affinitychromatography (at 4 to 6° C.) and ceramic hydroxyapatite (at roomtemperature). Buffer replacement steps after the rProtein A affinitychromatography purification and after the ceramic hydroxyapatitepurification were performed at 4 to 6° C. The culture supernatant wasapplied to MabSelectSuRe (GE Healthcare Bioscience, HiTrap column)equilibrated with PBS. After the entry of the whole culture supernatantin the column, the column was washed with at least twice the columnvolume of PBS. Next, fractions containing the antibody were collected byelution with a 2 M arginine hydrochloride solution (pH 4.0). Thefractions were subjected to buffer replacement with PBS by dialysis(Thermo Scientific, Slide-A-Lyzer Dialysis Cassette), and then diluted5-fold with 5 mM sodium phosphate/50 mM MES/pH 7.0 buffer, and theresulting antibody solution was applied to a ceramic hydroxyapatitecolumn (Bio-Rad Japan, Bio-Scale CHT Type-1 Hydroxyapatite Column)equilibrated with 5 mM NaPi/50 mM MES/30 mM NaCl/pH 7.0 buffer. Thefractions containing the antibody were collected by performing linearconcentration gradient elution with sodium chloride. The fractions weresubjected to buffer replacement with HBSor (25 mM histidine/5% sorbitol,pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer DialysisCassette). The fractions were concentrated using Centrifugal UF FilterDevice VIVASPIN20 (molecular weight cutoff: UF10K, Sartorius, at 4° C.),and adjusted to an IgG concentration of 2 mg/ml or more. Finally, thefractions were filtered using the Minisart-Plus filter (Sartorius) toobtain a purified sample.

Example 6

Evaluation of the in vitro activity of human chimeric anti-ALK2antibodies (cA2-15A and cA2-27D)

6)-1 Evaluation of the Antibodies by Luciferase Reporter Assay

The ALK2-mediated intracellular signaling inhibitory activity of each ofthe human chimeric antibodies prepared was analyzed using a BMP-specificluciferase reporter. HEPG2 cells were seeded into a 96-well white platefor luciferase assay (Corning) at 1×10⁴ cells/well, and culturedovernight in 10% FBS-containing DMEM medium under the conditions of 5%CO₂ at 37° C. On the following day, the HEPG2 cells were transfectedwith pGL4.26/Id1WT4F-luc (Genes Cells, 7, 949 (2002)), usingLipofectamine 2000 (Invitrogen). After 2.5 hours, the medium wasreplaced with fresh OPTI-MEM I (LifeTechnologies), and the cells werefurther cultured for 3 hours. Thereafter, the medium was replaced withOPTI-MEM I containing the serially diluted monoclonal antibody and 10ng/mL of BMP7 (Milteney), and the cells were further cultured overnight.On the following day, the luciferase activity was measured with theplate reader SpectraMaxM4 (Molecular Devices), using Dual-Glo LuciferaseAssay System (Promega).

The results are shown in FIG. 9. Each of the chimeric antibodies cA2-15Aand cA2-27D was confirmed to demonstrate an inhibitory activitycomparable to that of the rat monoclonal antibody A2-15A or A2-27D,respectively, upon the BMP7-induced BMP-specific luciferase activity.

6)-2 Evaluation of the Antibodies by BMP-Induced OsteoblastDifferentiation Assay

The inhibitory activity of each of the human chimeric antibodies againstendogenous ALK2-mediated intracellular signaling was analyzed based onthe effect upon the BMP-induced osteoblast differentiation of C2C12cells. C2C12 cells were seeded into a 96-well plate (Iwaki & Co., Ltd.)at 5×10³ cells/well, and cultured in 15% FBS-containing DMEM mediumovernight under the conditions of 5% C02 at 37° C. On the following day,the medium was replaced with fresh OPTI-MEM I (LifeTechnologies)containing the serially diluted monoclonal antibody and 2 ng/mL ofGDF2/BMP9 (Peprotech), and the cells were further cultured for 3 days.After the medium was removed from the C2C12 cells and the cells werewashed with PBS, the cells were treated with 50 μL/well of an ice-coldacetone:ethanol (1:1) solution for 1 minute, and further washed with PBSthree times. ALP activity was measured as an index of thedifferentiation into cells for forming osteoblasts. To measure ALPactivity, 100 μL/well of a substrate solution (0.1 M diethanolamine(Sigma-Aldrich)-HCl, pH 10.0, containing 1 mg/mL of 4-nitrophenylphosphate (Sigma-Aldrich) and 1 mM of MgCl₂) was added, and reacted for15 to 30 minutes at room temperature on an agitation shaker. Thereaction was stopped by the addition of 50 μL of 3M NaOH, and absorbanceat a wavelength of 405 nm was measured using the microplate readerSpectraMax M4 (Molecular Devices).

The results are shown in FIG. 10. It was confirmed that the chimericantibodies cA2-15A and cA2-27D suppressed the BMP-induceddifferentiation of the C2C12 cells into osteoblast-like cells in adose-dependent manner. These results show that the chimeric antibodiescA2-15A and cA2-27D are antibodies that suppress endogenous ALK2physiologically expressed by the C2C12 cells. Moreover, the inhibitoryactivity of each of the chimeric antibodies cA2-15A and cA2-27D wasconfirmed to be comparable to that of the rat monoclonal antibody A2-15Aor A2-27D, respectively.

Example 7

Design of humanized versions of anti-ALK2 antibodies 2-15A and A2-27D

7)-1 Design of Humanized hA2-15

7)-1-1 Molecular Modeling of the Variable Regions of A2-15A

The molecular modeling of the variable regions of A2-15A was performedusing a method known as homology modeling (Methods in Enzymology, 203,121-153, (1991)). The variable regions of A2-15A determined above werecompared with the primary sequences of human immunoglobulin variableregions registered in Protein Data Bank (three-dimensional structuresderived from X-ray crystal structures are available) (Nuc. Acid Res. 35,D301-D303 (2007)). As a result, 3KYM and 3S35 were selected as thosehaving the highest sequence identity to the heavy chain variable regionand the light chain variable region, respectively, of A2-15A. Thethree-dimensional structures of framework regions were prepared byobtaining a “framework model” by combining the coordinates of 3KYM and3S35 corresponding to the heavy chain and the light chain of A2-15A.Then, a representative conformation of each CDR was incorporated intothe framework model.

Finally, energy calculation for excluding disadvantageous interatomiccontact was performed, in order to obtain possible molecular models ofthe variable regions of A2-15A in terms of energy. The above-describedprocedures were performed using a commercially available proteinthree-dimensional structure analysis program, Discovery Studio(Accelrys, Inc.).

7)-1-2 Design of an Amino Acid Sequence for Humanized hA2-15A

The humanized hA2-15 was constructed using a method commonly known asCDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Anacceptor antibody was selected based on the amino acid identity inframework regions.

The sequences of the framework regions of A2-15 were compared with thesequences of framework regions of human subgroup consensus sequences. Asa result, the human γ chain subgroup 3 consensus sequence and the humanκ chain subgroup 4 consensus sequence defined by KABAT et al. (Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health ServiceNational Institutes of Health, Bethesda, Md. (1991)) were selected asacceptors due to their high sequence identity in framework regions. Theamino acid residues of the framework regions in the human γ chainsubgroup 3 consensus sequence and the human K chain subgroup 4 consensussequence were aligned with the amino acid residues of the frameworkregions of A2-15A to identify the numbers of amino acids differingtherebetween. The numbers of these residues were analyzed using thethree-dimensional model of A2-15A constructed above. Then, the donorresidues to be grafted onto the acceptors were selected in accordancewith the criteria provided by Queen et al. (Proc. Natl. Acad. Sci. USA86, 10029-10033 (1989)). Some donor residues thus selected weretransferred into the acceptor antibody to construct humanized hA2-15Asequences, as described in the following Examples.

7)-2 Humanization of A2-15A Heavy Chains

7)-2-1 Humanized hA2-15A-H1 Type Heavy Chain

A humanized hA2-15A heavy chain designed by substitution of amino acidnumber 35 (arginine) with glycine, amino acid number 38 (lysine) witharginine, amino acid number 61 (threonine) with glycine, amino acidnumber 94 (alanine) with serine, amino acid number 96 (serine) withasparagine, amino acid number 103 (aspartic acid) with asparagine, aminoacid number 107 (serine) with alanine, amino acid number 112 (threonine)with valine, and amino acid number 116 (threonine) with alanine in theheavy chain of chimeric cA2-15A shown in SEQ ID NO: 20 was designated as“humanized hA2-15A-H1 type heavy chain” (sometimes also referred to as“hA2-15A-H1”).

The amino acid sequence of the humanized hA2-15A-H1 type heavy chain isdescribed in SEQ ID NO: 28 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19, a sequence consisting of aminoacid numbers 20 to 142, and a sequence consisting of amino acid numbers143 to 472 in the amino acid sequence of SEQ ID NO: 28 correspond to thesignal sequence, the heavy chain variable region, and the heavy chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 28 is described in SEQ ID NO: 27 inthe Sequence Listing. A sequence consisting of nucleotide numbers 1 to57, a sequence consisting of nucleotide numbers 58 to 426, and asequence consisting of nucleotide numbers 427 to 1416 in the nucleotidesequence of SEQ ID NO: 27 encode the signal sequence, the heavy chainvariable region sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 27 and the aminoacid sequence of SEQ ID NO: 28 are also described in FIG. 15.

7)-2-2 Humanized hA2-15A-H4 Type Heavy Chain

A humanized hA2-15A heavy chain designed by substitution of amino acidnumber 35 (arginine) with glycine, amino acid number 38 (lysine) witharginine, amino acid number 61 (threonine) with glycine, amino acidnumber 103 (aspartic acid) with asparagine, and amino acid number 107(serine) with alanine in the heavy chain of chimeric cA2-15A shown inSEQ ID NO: 20 was designated as “humanized hA2-15A-H4 type heavy chain”(sometimes also referred to as “hA2-15A-H4”).

The amino acid sequence of the humanized hA2-15A-H4 type heavy chain isdescribed in SEQ ID NO: 30 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19, a sequence consisting of aminoacid numbers 20 to 142, and a sequence consisting of amino acid numbers143 to 472 in the amino acid sequence of SEQ ID NO: 30 correspond to thesignal sequence, the heavy chain variable region, and the heavy chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 30 is described in SEQ ID NO: 29 inthe Sequence Listing. A sequence consisting of nucleotide numbers 1 to57, a sequence consisting of nucleotide numbers 58 to 426, and asequence consisting of nucleotide numbers 427 to 1416 in the nucleotidesequence of SEQ ID NO: 29 encode the signal sequence, the heavy chainvariable region sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 29 and the aminoacid sequence of SEQ ID NO: 30 are also described in FIG. 16.

7)-3 Humanization of A2-15A Light Chain

7)-3-1 Humanized hA2-15A-L1 Type Light Chain

A humanized hA2-15A light chain designed by the substitution of aminoacid number 24 (leucine) with methionine, amino acid number 29 (alanine)with aspartic acid, a position (missing residue) between amino acidnumbers 29 and 30 with serine, amino acid number 36 (glutamine) withglutamic acid, amino acid number 41 (serine) with asparagine, amino acidnumber 66 (lysine) with proline, amino acid number 81 (isoleucine) withvaline, amino acid number 83 (alanine) with aspartic acid, amino acidnumber 99 (asparagine) with serine, amino acid number 100 (proline) withserine, amino acid number 101 (valine) with leucine, amino acid number104 (aspartic acid) with glutamic acid, amino acid number 106(isoleucine) with valine, amino acid number 108 (threonine) with valine,amino acid number 123 (alanine) with glutamine, amino acid number 127(leucine) with valine, and amino acid number 129 (leucine) withisoleucine in the light chain of chimeric cA2-15A shown in SEQ ID NO: 22was designated as “humanized hA2-15A-L1 type light chain” (sometimesalso referred to as “hA2-15A-L1”).

The amino acid sequence of the humanized hA2-15A-L1 type light chain isdescribed in SEQ ID NO: 32 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20, a sequence consisting of aminoacid numbers 21 to 133, and a sequence consisting of amino acid numbers134 to 238 in the amino acid sequence of SEQ ID NO: 32 correspond to thesignal sequence, the light chain variable region, and the light chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 32 is described in SEQ ID NO: 31 inthe Sequence Listing. A sequence consisting of nucleotide numbers 26 to85, a sequence consisting of nucleotide numbers 86 to 424, and asequence consisting of nucleotide numbers 425 to 739 in the nucleotidesequence of SEQ ID NO: 31 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 31 and the aminoacid sequence of SEQ ID NO: 32 are also described in FIG. 17.

7)-3-2 Humanized hA2-15A-L4 Type Light Chain

A humanized hA2-15A light chain designed by the substitution of aminoacid number 29 (alanine) with aspartic acid, a position (missingresidue) between amino acid numbers 29 and 30 with serine, amino acidnumber 36 (glutamine) with glutamic acid, amino acid number 41 (serine)with asparagine, amino acid number 99 (asparagine) with serine, aminoacid number 100 (proline) with serine, amino acid number 108 (threonine)with valine, and amino acid number 123 (alanine) with glutamine in thelight chain of chimeric cA2-15A shown in SEQ ID NO: 22 was designated as“humanized hA2-15A-L4 type light chain” (sometimes also referred to as“hA2-15A-L4”).

The amino acid sequence of the humanized hA2-15A-L4 type light chain isdescribed in SEQ ID NO: 34 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20, a sequence consisting of aminoacid numbers 21 to 133, and a sequence consisting of amino acid numbers134 to 238 in the amino acid sequence of SEQ ID NO: 34 correspond to thesignal sequence, the light chain variable region, and the light chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 34 is described in SEQ ID NO: 33 inthe Sequence Listing. A sequence consisting of nucleotide numbers 26 to85, a sequence consisting of nucleotide numbers 86 to 424, and asequence consisting of nucleotide numbers 425 to 739 in the nucleotidesequence of SEQ ID NO: 33 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 33 and the aminoacid sequence of SEQ ID NO: 34 are also described in FIG. 18.

7)-3-3 Humanized hA2-15A-L6 Type Light Chain

A humanized hA2-15A light chain designed by the substitution of aminoacid number 29 (alanine) with aspartic acid, a position (missingresidue) between amino acid numbers 29 and 30 with serine, amino acidnumber 36 (glutamine) with glutamic acid, amino acid number 41 (serine)with asparagine, amino acid number 79 (serine) with glutamine, aminoacid number 99 (asparagine) with serine, amino acid number 100 (proline)with serine, amino acid number 108 (threonine) with valine, and aminoacid number 123 (alanine) with glutamine in the light chain of chimericcA2-15A shown in SEQ ID NO: 22 was designated as “humanized hA2-15A-L6type light chain” (sometimes also referred to as “hA2-15A-L6”).

The amino acid sequence of the humanized hA2-15A-L6 type light chain isdescribed in SEQ ID NO: 36 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20, a sequence consisting of aminoacid numbers 21 to 133, and a sequence consisting of amino acid numbers134 to 238 in the amino acid sequence of SEQ ID NO: 36 correspond to thesignal sequence, the light chain variable region, and the light chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 36 is described in SEQ ID NO: 35 inthe Sequence Listing. A sequence consisting of nucleotide numbers 26 to85, a sequence consisting of nucleotide numbers 86 to 424, and asequence consisting of nucleotide numbers 425 to 739 in the nucleotidesequence of SEQ ID NO: 35 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 35 and the aminoacid sequence of SEQ ID NO: 36 are also described in FIG. 19.

7)-3-4 Humanized hA2-15A-L7 Type Light Chain

A humanized hA2-15A light chain designed by the substitution of aminoacid number 29 (alanine) with aspartic acid, a position (missingresidue) between amino acid numbers 29 and 30 with serine, amino acidnumber 36 (glutamine) with glutamic acid, amino acid number 41 (serine)with asparagine, amino acid number 70 (leucine) with alanine, amino acidnumber 99 (asparagine) with serine, amino acid number 100 (proline) withserine, amino acid number 108 (threonine) with valine, and amino acidnumber 123 (alanine) with glutamine in the light chain of chimericcA2-15A shown in SEQ ID NO: 22 was designated as “humanized hA2-15A-L7type light chain” (sometimes also referred to as “hA2-15A-L7”).

The amino acid sequence of the humanized hA2-15A-L7 type light chain isdescribed in SEQ ID NO: 38 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20, a sequence consisting of aminoacid numbers 21 to 133, and a sequence consisting of amino acid numbers134 to 238 in the amino acid sequence of SEQ ID NO: 38 correspond to thesignal sequence, the light chain variable region, and the light chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 38 is described in SEQ ID NO: 37 inthe Sequence Listing. A sequence consisting of nucleotide numbers 26 to85, a sequence consisting of nucleotide numbers 86 to 424, and asequence consisting of nucleotide numbers 425 to 739 in the nucleotidesequence of SEQ ID NO: 37 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 37 and the aminoacid sequence of SEQ ID NO: 38 are also described in FIG. 20.

7)-4 Design of Humanized hA2-15A by Combinations of Heavy Chains andLight Chains

An antibody consisting of the humanized hA2-15A-H1 type heavy chain andthe humanized hA2-15A-L1 type light chain was designed and designated as“humanized hA2-15A-H1/L1” (sometimes also referred to as“hA2-15A-H1/L1”). An antibody consisting of the humanized hA2-15A-H1type heavy chain and the humanized hA2-15A-L4 type light chain wasdesigned and designated as “humanized hA2-15A-H1/L4” (sometimes alsoreferred to as “hA2-15A-H1/L4”). An antibody consisting of the humanizedhA2-15A-H4 type heavy chain and the humanized hA2-15A-L1 type lightchain was designed and designated as “humanized hA2-15A-H4/L1”(sometimes also referred to as “hA2-15A-H4/L1”). An antibody consistingof the humanized hA2-15A-H4 type heavy chain and the humanizedhA2-15A-L4 type light chain was designed and designated as “humanizedhA2-15A-H4/L4” (sometimes also referred to as “hA2-15A-H4/L4”). Anantibody consisting of the humanized hA2-15A-H4 type heavy chain and thehumanized hA2-15A-L6 type light chain was designed and designated as“humanized hA2-15A-H4/L6” (sometimes also referred to as“hA2-15A-H4/L6”). An antibody consisting of the humanized hA2-15A-H4type heavy chain and the humanized hA2-15A-L7 type light chain wasdesigned and designated as “humanized hA2-15A-H4/L7” (sometimes alsoreferred to as “hA2-15A-H4/L7”). The antibodies designed above can beprepared in accordance with Example 8, and evaluated in accordance withExamples 2 and 4.

7)-5 Design of Humanized hA2-27D

7)-5-1 Molecular Modeling of the Variable Regions of A2-27D

The molecular modeling of the variable regions of A2-27D was performedusing a method known as homology modeling (Methods in Enzymology, 203,121-153, (1991)). The variable regions of A2-27D determined above werecompared with the primary sequences of human immunoglobulin variableregions registered in Protein Data Bank (three-dimensional structuresderived from X-ray crystal structures are available) (Nuc. Acid Res. 35,D301-D303 (2007)). As a result, 3EYQ and 419W were selected as thosehaving the highest sequence identity to the heavy chain variable regionand the light chain variable region, respectively, of A2-27D. Thethree-dimensional structures of framework regions were prepared byobtaining a “framework model” by combining the coordinates of 3EYQ and419W corresponding to the heavy chain and the light chain of A2-27D.Then, a representative conformation of each CDR was incorporated intothe framework model.

Finally, energy calculation for excluding disadvantageous interatomiccontact was performed, in order to obtain possible molecular models ofthe variable regions of A2-27D in terms of energy. The above-describedprocedures were performed using a commercially available proteinthree-dimensional structure analysis program, Discovery Studio(Accelrys, Inc.).

7)-5-2 Design of an Amino Acid Sequence for Humanized hA2-27D

The humanized hA2-27D was constructed using a method commonly known asCDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Anacceptor antibody was selected based on the amino acid identity inframework regions.

The sequences of the framework regions of A2-27D were compared with thesequences of framework regions of human subgroup consensus sequences. Asa result, the human γ chain subgroup 3 consensus sequence and the humanκ chain subgroup 4 consensus sequence defined by KABAT et al. (Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health ServiceNational Institutes of Health, Bethesda, Md. (1991)) were selected asacceptors due to their high sequence identity in framework regions. Theamino acid residues of the framework regions in the human γ chainsubgroup 3 consensus sequence and the human K chain subgroup 3 consensussequence were aligned with the amino acid residues of the frameworkregions of A2-27D to identify the numbers of amino acids differingtherebetween. The numbers of these residues were analyzed using thethree-dimensional model of A2-27D constructed above. Then, the donorresidues to be grafted onto the acceptors were selected in accordancewith the criteria provided by Queen et al. (Proc. Natl. Acad. Sci. USA86, 10029-10033 (1989)). Some donor residues thus selected weretransferred into the acceptor antibody to construct humanized hA2-27Dsequences, as described in the following Examples.

7)-6 Humanization of A2-27D Heavy Chains

7)-6-1 Humanized hA2-27D-H1 Type Heavy Chain

A humanized hA2-15A heavy chain designed by the substitution of aminoacid number 35 (arginine) with glycine, amino acid number 38 (lysine)with arginine, amino acid number 42 (leucine) with alanine, amino acidnumber 56 (isoleucine) with valine, amino acid number 68 (alanine) withserine, amino acid number 94 (alanine) with serine, amino acid number 95(arginine) with lysine, amino acid number 103 (threonine) withasparagine, amino acid number 107 (serine) with alanine, amino acidnumber 112 (leucine) with valine, amino acid number 117 (alanine) witharginine, amino acid number 132 (proline) with glutamine, and amino acidnumber 135 (valine) with leucine in the heavy chain of chimeric cA2-27Dshown in SEQ ID NO: 24 was designated as “humanized hA2-27D-H1 typeheavy chain” (sometimes also referred to as “hA2-27D-H1”).

The amino acid sequence of the humanized hA2-27D-H1 type heavy chain isdescribed in SEQ ID NO: 40 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19, a sequence consisting of aminoacid numbers 20 to 140, and a sequence consisting of amino acid numbers141 to 470 in the amino acid sequence of SEQ ID NO: 40 correspond to thesignal sequence, the heavy chain variable region, and the heavy chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 40 is described in SEQ ID NO: 39 inthe Sequence Listing. A sequence consisting of nucleotide numbers 1 to57, a sequence consisting of nucleotide numbers 58 to 420, and asequence consisting of nucleotide numbers 421 to 1410 in the nucleotidesequence of SEQ ID NO: 39 encode the signal sequence, the heavy chainvariable region sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 39 and the aminoacid sequence of SEQ ID NO: 40 are also described in FIG. 21.

7)-6-2 Humanized hA2-27D-H2 Type Heavy Chain

A humanized hA2-27D heavy chain designed by the substitution of aminoacid number 35 (arginine) with glycine, amino acid number 38 (lysine)with arginine, amino acid number 42 (leucine) with alanine, amino acidnumber 68 (alanine) with serine, amino acid number 94 (alanine) withserine, amino acid number 95 (arginine) with lysine, amino acid number103 (threonine) with asparagine, amino acid number 107 (serine) withalanine, amino acid number 112 (leucine) with valine, amino acid number132 (proline) with glutamine, and amino acid number 135 (valine) withleucine in the heavy chain of chimeric cA2-27D shown in SEQ ID NO: 24was designated as “humanized hA2-27D-H2 type heavy chain” (sometimesalso referred to as “hA2-27D-H2”).

The amino acid sequence of the humanized hA2-27D-H2 type heavy chain isdescribed in SEQ ID NO: 42 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19, a sequence consisting of aminoacid numbers 20 to 140, and a sequence consisting of amino acid residues141 to 470 in the amino acid sequence of SEQ ID NO: 42 correspond to thesignal sequence, the heavy chain variable region, and the heavy chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 42 is described in SEQ ID NO: 41 inthe Sequence Listing. A sequence consisting of nucleotide numbers 1 to57, a sequence consisting of nucleotide numbers 58 to 420, and asequence consisting of nucleotide numbers 421 to 1410 in the nucleotidesequence of SEQ ID NO: 41 encode the signal sequence, the heavy chainvariable region sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 41 and the aminoacid sequence of SEQ ID NO: 42 are also described in FIG. 22.

7)-6-3 Humanized hA2-27D-H3 Type Heavy Chain

A humanized hA2-27D heavy chain designed by the substitution of aminoacid number 35 (arginine) with glycine, amino acid number 38 (lysine)with arginine, amino acid number 42 (leucine) with alanine, amino acidnumber 94 (alanine) with serine, amino acid number 95 (arginine) withlysine, amino acid number 103 (threonine) with asparagine, amino acidnumber 107 (serine) with alanine, amino acid number 112 (leucine) withvaline, amino acid number 132 (proline) with glutamine, and amino acidnumber 135 (valine) with leucine in the heavy chain of chimeric cA2-27Dshown in SEQ ID NO: 24 was designated as “humanized hA2-27D-H3 typeheavy chain” (sometimes also referred to as “hA2-27D-H3”).

The amino acid sequence of the humanized hA2-27D-H3 type heavy chain isdescribed in SEQ ID NO: 44 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19, a sequence consisting of aminoacid numbers 20 to 140, and a sequence consisting of amino acid numbers141 to 470 in the amino acid sequence of SEQ ID NO: 44 correspond to thesignal sequence, the heavy chain variable region, and the heavy chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 44 is described in SEQ ID NO: 43 inthe Sequence Listing. A sequence consisting of nucleotide numbers 1 to57, a sequence consisting of nucleotides 58 to 420, and a sequenceconsisting of nucleotide numbers 421 to 1410 in the nucleotide sequenceof SEQ ID NO: 43 encode the signal sequence, the heavy chain variableregion sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 43 and the aminoacid sequence of SEQ ID NO: 44 are also described in FIG. 23.

7)-6-4 Humanized hA2-27D-H4 Type Heavy Chain

A humanized hA2-27D heavy chain designed by the substitution of aminoacid number 35 (arginine) with glycine, amino acid number 38 (lysine)with arginine, amino acid number 42 (leucine) with alanine, amino acidnumber 94 (alanine) with serine, amino acid number 95 (arginine) withlysine, amino acid number 103 (threonine) with asparagine, amino acidnumber 107 (serine) with alanine, and amino acid number 135 (valine)with leucine in the heavy chain of chimeric cA2-27D shown in SEQ ID NO:24 was designated as “humanized hA2-27D-H4 type heavy chain” (sometimesalso referred to as “hA2-27D-H4”).

The amino acid sequence of the humanized hA2-27D-H4 type heavy chain isdescribed in SEQ ID NO: 46 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19, a sequence consisting of aminoacid numbers 20 to 140, and a sequence consisting of amino acid numbers141 to 470 in the amino acid sequence of SEQ ID NO: 46 correspond to thesignal sequence, the heavy chain variable region, and the heavy chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 46 is described in SEQ ID NO: 45 inthe Sequence Listing. A sequence consisting of nucleotide numbers 1 to57, a sequence consisting of nucleotide numbers 58 to 420, and asequence consisting of nucleotide numbers 421 to 1410 in the nucleotidesequence of SEQ ID NO: 45 encode the signal sequence, the heavy chainvariable region sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 45 and the aminoacid sequence of SEQ ID NO: 46 are also described in FIG. 24.

7)-6-5 Humanized hA2-27D-H5 Type Heavy Chain

A humanized hA2-27D heavy chain designed by the substitution of aminoacid number 35 (arginine) with glycine, amino acid number 38 (lysine)with arginine, amino acid number 42 (leucine) with alanine, amino acidnumber 95 (arginine) with lysine, amino acid number 103 (threonine) withasparagine, and amino acid number 135 (valine) with leucine in the heavychain of chimeric cA2-27D shown in SEQ ID NO: 24 was designated as“humanized hA2-27D-H5 type heavy chain” (sometimes also referred to as“hA2-27D-H5”).

The amino acid sequence of the humanized hA2-27D-H5 type heavy chain isdescribed in SEQ ID NO: 48 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19, a sequence consisting of aminoacid numbers 20 to 140, and a sequence consisting of amino acid numbers141 to 470 in the amino acid sequence of SEQ ID NO: 48 correspond to thesignal sequence, the heavy chain variable region, and the heavy chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 48 is described in SEQ ID NO: 47 inthe Sequence Listing. A sequence consisting of nucleotide numbers 1 to57, a sequence consisting of nucleotide numbers 58 to 420, and asequence consisting of nucleotide numbers 421 to 1410 in the nucleotidesequence of SEQ ID NO: 47 encode the signal sequence, the heavy chainvariable region sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 47 and the aminoacid sequence of SEQ ID NO: 48 are also described in FIG. 25.

7)-7 Humanization of A2-27D Light Chains

7)-7-1 Humanized hA2-27D-L1 Type Light Chain

A humanized hA2-27D light chain designed by the substitution of aminoacid number 29 (threonine) with glycine, amino acid number 31(methionine) with leucine, amino acid number 32 (alanine) with serine,amino acid number 33 (alanine) with leucine, amino acid number 38(lysine) with arginine, amino acid number 39 (valine) with alanine,amino acid number 42 (asparagine) with serine, amino acid number 59(serine) with proline, amino acid number 61 (alanine) with glutamine,amino acid number 62 (serine) with alanine, amino acid number 64(lysine) with arginine, amino acid number 66 (tryptophan) with leucine,amino acid number 77 (valine) with isoleucine, amino acid number 79(asparagine) with aspartic acid, amino acid number 89 (serine) withaspartic acid, amino acid number 90 (tyrosine) with phenylalanine, aminoacid number 91 (serine) with threonine, amino acid number 93 (alanine)with threonine, amino acid number 96 (serine) with arginine, amino acidnumber 97 (methionine) with leucine, amino acid number 99 (alanine) withproline, amino acid number 102 (valine) with phenylalanine, amino acidnumber 104 (threonine) with valine, amino acid number 120 (alanine) withglutamine, amino acid number 124 (leucine) with valine, and amino acidnumber 126 (leucine) with isoleucine in the light chain of chimericcA2-27D shown in SEQ ID NO: 26 was designated as “humanized hA2-27D-L1type light chain” (sometimes also referred to as “hA2-27D-L 1”).

The amino acid sequence of the humanized hA2-27D-L1 type light chain isdescribed in SEQ ID NO: 50 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20, a sequence consisting of aminoacid numbers 21 to 129, and a sequence consisting of amino acid numbers130 to 234 in the amino acid sequence of SEQ ID NO: 50 correspond to thesignal sequence, the light chain variable region, and the light chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 50 is described in SEQ ID NO: 49 inthe Sequence Listing. A sequence consisting of nucleotide numbers 26 to85, a sequence consisting of nucleotide numbers 86 to 412, and asequence consisting of nucleotide numbers 413 to 727 in the nucleotidesequence of SEQ ID NO: 49 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 49 and the aminoacid sequence of SEQ ID NO: 50 are also described in FIG. 26.

7)-7-2 Humanized hA2-27D-L2 Type Light Chain

A humanized hA2-27D light chain designed by the substitution of aminoacid number 29 (threonine) with glycine, amino acid number 31(methionine) with leucine, amino acid number 32 (alanine) with serine,amino acid number 33 (alanine) with leucine, amino acid number 38(lysine) with arginine, amino acid number 39 (valine) with alanine,amino acid number 42 (asparagine) with serine, amino acid number 59(serine) with proline, amino acid number 61 (alanine) with glutamine,amino acid number 64 (lysine) with arginine, amino acid number 79(asparagine) with aspartic acid, amino acid number 89 (serine) withaspartic acid, amino acid number 90 (tyrosine) with phenylalanine, aminoacid number 91 (serine) with threonine, amino acid number 93 (alanine)with threonine, amino acid number 96 (serine) with arginine, amino acidnumber 97 (methionine) with leucine, amino acid number 99 (alanine) withproline, amino acid number 102 (valine) with phenylalanine, amino acidnumber 104 (threonine) with valine, amino acid number 120 (alanine) withglutamine, amino acid number 124 (leucine) with valine, and amino acidnumber 126 (leucine) with isoleucine in the light chain of chimericcA2-27D shown in SEQ ID NO: 26 was designated as “humanized hA2-27D-L2type light chain” (sometimes also referred to as “hA2-27D-L2”).

The amino acid sequence of the humanized hA2-27D-L2 type light chain isdescribed in SEQ ID NO: 52 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20, a sequence consisting of aminoacid numbers 21 to 129, and a sequence consisting of amino acid numbers130 to 234 in the amino acid sequence of SEQ ID NO: 52 correspond to thesignal sequence, the light chain variable region, and the light chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 52 is described in SEQ ID NO: 51 inthe Sequence Listing. A sequence consisting of nucleotide numbers 26 to85, a sequence consisting of nucleotide numbers 86 to 412, and asequence consisting of nucleotide numbers 413 to 727 in the nucleotidesequence of SEQ ID NO: 51 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 51 and the aminoacid sequence of SEQ ID NO: 52 are also described in FIG. 27.

7)-7-3 Humanized hA2-27D-L3 Type Light Chain

A humanized hA2-27D light chain designed by the substitution of aminoacid number 29 (threonine) with glycine, amino acid number 31(methionine) with leucine, amino acid number 32 (alanine) with serine,amino acid number 33 (alanine) with leucine, amino acid number 38(lysine) with arginine, amino acid number 39 (valine) with alanine,amino acid number 42 (asparagine) with serine, amino acid number 59(serine) with proline, amino acid number 64 (lysine) with arginine,amino acid number 79 (asparagine) with aspartic acid, amino acid number89 (serine) with aspartic acid, amino acid number 91 (serine) withthreonine, amino acid number 93 (alanine) with threonine, amino acidnumber 96 (serine) with arginine, amino acid number 97 (methionine) withleucine, amino acid number 99 (alanine) with proline, amino acid number102 (valine) with phenylalanine, amino acid number 124 (leucine) withvaline, and amino acid number 126 (leucine) with isoleucine in the lightchain of chimeric cA2-27D shown in SEQ ID NO: 26 was designated as“humanized hA2-27D-L3 type light chain” (sometimes also referred to as“hA2-27D-L3”).

The amino acid sequence of the humanized hA2-27D-L3 type light chain isdescribed in SEQ ID NO: 54 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20, a sequence consisting of aminoacid numbers 21 to 129, and a sequence consisting of amino acid numbers130 to 234 in the amino acid sequence of SEQ ID NO: 54 correspond to thesignal sequence, the light chain variable region, and the light chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 54 is described in SEQ ID NO: 53 inthe Sequence Listing. A sequence consisting of nucleotide numbers 26 to85, a sequence consisting of nucleotide numbers 86 to 412, and asequence consisting of nucleotide numbers 413 to 727 in the nucleotidesequence of SEQ ID NO: 53 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 53 and the aminoacid sequence of SEQ ID NO: 54 are also described in FIG. 28.

7)-7-4 Humanized hA2-27D-L4 Type Light Chain

A humanized hA2-27D light chain was designed by the substitution ofamino acid number 29 (threonine) with glycine, amino acid number 32(alanine) with serine, amino acid number 38 (lysine) with arginine,amino acid number 42 (asparagine) with serine, amino acid number 59(serine) with proline, amino acid number 64 (lysine) with arginine,amino acid number 79 (asparagine) with aspartic acid, amino acid number89 (serine) with aspartic acid, amino acid number 91 (serine) withthreonine, amino acid number 93 (alanine) with threonine, amino acidnumber 96 (serine) with arginine, amino acid number 99 (alanine) withproline, and amino acid number 102 (valine) with phenylalanine in thelight chain of chimeric cA2-27D shown in SEQ ID NO: 26 was designated as“humanized hA2-27D-L4 type light chain” (sometimes also referred to as“hA2-27D-L4”).

The amino acid sequence of the humanized hA2-27D-L4 type light chain isdescribed in SEQ ID NO: 56 in the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20, a sequence consisting of aminoacid numbers 21 to 129, and a sequence consisting of amino acid numbers130 to 234 in the amino acid sequence of SEQ ID NO: 56 correspond to thesignal sequence, the light chain variable region, and the light chainconstant region, respectively. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 56 is described in SEQ ID NO: 55 inthe Sequence Listing. A sequence consisting of nucleotide numbers 26 to85, a sequence consisting of nucleotide numbers 86 to 412, and asequence consisting of nucleotide numbers 413 to 727 in the nucleotidesequence of SEQ ID NO: 55 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 55 and the aminoacid sequence of SEQ ID NO: 56 are also described in FIG. 29.

7)-7-5 Humanized hA2-27D-L5 Type Light Chain

A humanized hA2-27D light chain designed by the substitution of aminoacid number 29 (threonine) with glycine, amino acid number 32 (alanine)with serine, amino acid number 38 (lysine) with arginine, amino acidnumber 91 (serine) with threonine, amino acid number 93 (alanine) withthreonine, amino acid number 96 (serine) with arginine, amino acidnumber 99 (alanine) with proline, and amino acid number 102 (valine)with phenylalanine in the light chain of chimeric cA2-27D shown in SEQID NO: 26 was designated as “humanized hA2-27D-L5 type light chain”(sometimes also referred to as “hA2-27D-L5”). The amino acid sequence ofthe humanized hA2-27D-L5 type light chain is described in SEQ ID NO: 58in the Sequence Listing. A sequence consisting of amino acid numbers 1to 20, a sequence consisting of amino acid numbers 21 to 129, and asequence consisting of amino acid numbers 130 to 234 in the amino acidsequence of SEQ ID NO: 58 correspond to the signal sequence, the lightchain variable region, and the light chain constant region,respectively. The nucleotide sequence encoding the amino acid sequenceof SEQ ID NO: 58 is described in SEQ ID NO: 57 in the Sequence Listing.A sequence consisting of nucleotide numbers 26 to 85, a sequenceconsisting of nucleotide numbers 86 to 412, and a sequence consisting ofnucleotide numbers 413 to 727 in the nucleotide sequence of SEQ ID NO:57 encode the signal sequence, the light chain variable region sequence,and the light chain constant region sequence, respectively. Thenucleotide sequence of SEQ ID NO: 57 and the amino acid sequence of SEQID NO: 58 are also described in FIG. 30.

7)-8 Design of Humanized hA2-27D by Combinations of Heavy Chains andLight Chains

An antibody consisting of the humanized hA2-27D-H1 type heavy chain andthe humanized hA2-27D-L1 type light chain was designed and designated as“humanized hA2-27D-H1/L1” (sometimes also referred to as“hA2-27D-H1/L1”). An antibody consisting of the humanized hA2-27D-H1type heavy chain and the humanized hA2-27D-L2 type light chain wasdesigned and designated as “humanized hA2-27D-H1/L2” (sometimes alsoreferred to as “hA2-27D-H1/L2”). An antibody consisting of the humanizedhA2-27D-H1 type heavy chain and the humanized hA2-27D-L3 type lightchain was designed and designated as “humanized hA2-27D-H1/L3”(sometimes also referred to as “hA2-27D-H1/L3”). An antibody consistingof the humanized hA2-27D-H2 type heavy chain and the humanizedhA2-27D-L1 type light chain was designed and designated as “humanizedhA2-27D-H2/L1” (sometimes also referred to as “hA2-27D-H2/L1”). Anantibody consisting of the humanized hA2-27D-H2 type heavy chain and thehumanized hA2-27D-L2 type light chain was designed and designated as“humanized hA2-27D-H2/L2” (sometimes also referred to as“hA2-27D-H2/L2”). An antibody consisting of the humanized hA2-27D-H2type heavy chain and the humanized hA2-27D-L3 type light chain wasdesigned and designated as “humanized hA2-27D-H2/L3” (sometimes alsoreferred to as “hA2-27D-H2/L3”). An antibody consisting of the humanizedhA2-27D-H3 type heavy chain and the humanized hA2-27D-L1 type lightchain was designed and designated as “humanized hA2-27D-H3/L1”(sometimes also referred to as “hA2-27D-H3/L1”). An antibody consistingof the humanized hA2-27D-H3 type heavy chain and the humanizedhA2-27D-L2 type light chain was designed and designated as “humanizedhA2-27D-H3/L2” (sometimes also referred to as “hA2-27D-H3/L2”). Anantibody consisting of the humanized hA2-27D-H3 type heavy chain and thehumanized hA2-27D-L3 type light chain was designed and designated as“humanized hA2-27D-H3/L3” (sometimes also referred to as“hA2-27D-H3/L3”). An antibody consisting of the humanized hA2-27D-H3type heavy chain and the humanized hA2-27D-L4 type light chain wasdesigned and designated as “humanized hA2-27D-H3/L4” (sometimes alsoreferred to as “hA2-27D-H3/L4”). An antibody consisting of the humanizedhA2-27D-H4 type heavy chain and the humanized hA2-27D-L3 type lightchain was designed and designated as “humanized hA2-27D-H4/L3”(sometimes also referred to as “hA2-27D-H4/L3”). An antibody consistingof the humanized hA2-27D-H4 type heavy chain and the humanizedhA2-27D-L4 type light chain was designed and designated as “humanizedhA2-27D-H4/L4” (sometimes also referred to as “hA2-27D-H4/L4”). Anantibody consisting of the humanized hA2-27D-H4 type heavy chain and thehumanized hA2-27D-L5 type light chain was designed and designated as“humanized hA2-27D-H4/L5” (sometimes also referred to as“hA2-27D-H4/L5”). An antibody consisting of the humanized hA2-27D-H5type heavy chain and the humanized hA2-27D-L4 type light chain wasdesigned and designated as “humanized hA2-27D-H5/L4” (sometimes alsoreferred to as “hA2-27D-H5/L4”). The antibodies designed as above can beprepared in accordance with Example 8, and evaluated in accordance withExamples 2 and 4.

Example 8 Construction of Humanized A2-15A Antibody and Humanized A2-27DAntibody Expression Vectors and Preparation of the Antibodies

8)-1 Construction of Humanized A2-15A Heavy Chain Expression Vector

8)-1-1 Construction of Humanized hA2-15A-H1 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-15A-H1 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 443of the nucleotide sequence of humanized hA2-15A-H1 shown in SEQ ID NO:27 was synthesized (GeneArt Artificial Gene Synthesis Service). The DNAfragment containing a humanized hA2-15A-H1 variable region-encoding DNAsequence was amplified using the synthesized DNA fragment as a template,KOD-Plus- (Toyobo Co., Ltd., Japan), and a primer set given below, andinserted at the restriction enzyme BlpI-cleaved site of the chimeric andhumanized antibody IgG1 type heavy chain expression vector pCMA-G1 usingIn-Fusion HD PCR cloning kit (Clontech Laboratories, Inc.) to constructa humanized hA2-15A-H1 expression vector. The obtained expression vectorwas designated as “pCMA-G1/hA2-15A-H1”.

Primer set (EG-Inf-F; SEQ ID NO: 99) 5′-AGCTCCCAGATGGGTGCTGAGC-3′(EG1-Inf-R; SEQ ID NO: 100) 5′-GGGCCCTTGGTGGAGGCTGAGC-3′

8)-1-2 Construction of Humanized hA2-15A-H4 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-15A-H4 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 443of the nucleotide sequence of humanized hA2-15A-H4 shown in SEQ ID NO:29 was synthesized (GeneArt Artificial Gene Synthesis Service). Ahumanized hA2-15A-H4 expression vector was constructed in the same wayas in Example 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-15A-H4”.

8)-2 Construction of Humanized A2-15A Light Chain Expression Vector

8)-2-1 Construction of Humanized hA2-15A-L1 Type Light Chain ExpressionVector

A DNA fragment containing a sequence encoding humanized hA2-15A-L1, ofSEQ ID NO: 31, was synthesized (GeneArt Gene Synthesis Service). The DNAfragment containing a sequence encoding humanized hA2-15A-L1 wasamplified using the synthesized DNA fragment as a template, KOD-Plus-(Toyobo Co., Ltd.), and a primer set given below, and inserted at thesite from which the sequence encoding a K chain secretory signal and ahuman K chain constant region was removed by the digestion of theexpression vector pCMA-LK with restriction enzymes XbaI and PmeI, usingIn-Fusion HD PCR cloning kit (Clontech Laboratories, Inc.) to constructa humanized hA2-15A-L1 expression vector. The obtained expression vectorwas designated as “pCMA/hA2-15A-L1”.

Primer set (CM-inf-F; SEQ ID NO: 101) 5′-CCAGCCTCCGGACTCTAGAGCCACC-3′(CM-inf-R; SEQ ID NO: 102) 5′-AGTTAGCCTCCCCCGTTTAAACTC-3′

8)-2-2 Construction of Humanized hA2-15A-L4 Type Light Chain ExpressionVector

A DNA fragment containing a sequence encoding humanized hA2-15A-L4, ofSEQ ID NO: 33, was synthesized (GeneArt Gene Synthesis Service). Ahumanized hA2-15A-L4 expression vector was constructed in the same wayas in Example 8)-2-1. The obtained expression vector was designated as“pCMA/hA2-15A-L4”.

8)-2-3 Construction of Humanized hA2-15A-L6 Type Light Chain ExpressionVector

A DNA fragment containing a sequence encoding humanized hA2-15A-L6, asshown in SEQ ID NO: 35, was synthesized (GeneArt Gene SynthesisService). A humanized hA2-15A-L6 expression vector was constructed inthe same way as in Example 8)-2-1. The obtained expression vector wasdesignated as “pCMA/hA2-15A-L6”.

8)-2-4 Construction of Humanized hA2-15A-L7 Type Light Chain ExpressionVector

A DNA fragment containing a sequence encoding humanized hA2-15A-L7, asshown in SEQ ID NO: 37, was synthesized (GeneArt Gene SynthesisService). A humanized hA2-15A-L7 expression vector was constructed inthe same way as in Example 8)-2-1. The obtained expression vector wasdesignated as “pCMA/hA2-15A-L7”.

8)-3 Construction of Humanized A2-27D Heavy Chain Expression Vector

8)-3-1 Construction of Humanized hA2-27D-H1 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-27D-H1 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 437of the nucleotide sequence of humanized hA2-27D-H1 of SEQ ID NO: 39 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-27D-H1 expression vector was constructed in the same way as inExample 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-27D-H1”.

8)-3-2 Construction of Humanized hA2-27D-H2 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-27D-H2 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 437of the nucleotide sequence of humanized hA2-27D-H2 of SEQ ID NO: 41 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-27D-H2 expression vector was constructed in the same way as inExample 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-27D-H2”.

8)-3-3 Construction of Humanized hA2-27D-H3 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-27D-H3 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 437of the nucleotide sequence of humanized hA2-27D-H3 of SEQ ID NO: 43 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-27D-H3 expression vector was constructed in the same way as inExample 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-27D-H3”.

8)-3-4 Construction of Humanized hA2-27D-H4 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-27D-H4 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 437of the nucleotide sequence of humanized hA2-27D-H4 of SEQ ID NO: 45 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-27D-H4 expression vector was constructed in the same way as inExample 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-27D-H4”.

8)-3-5 Construction of Humanized hA2-27D-H5 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-27D-H5 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 437of the nucleotide sequence of humanized hA2-27D-H5 of SEQ ID NO: 47 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-27D-H5 expression vector was constructed in the same way as inExample 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-27D-H5”.

8)-4 Construction of Humanized A2-27D Light Chain Expression Vector

8)-4-1 Construction of Humanized hA2-27D-L 1 Type Light Chain ExpressionVector

A DNA fragment containing a sequence encoding humanized hA2-27D-L1, asshown in SEQ ID NO: 49, was synthesized (GeneArt Gene SynthesisService). A humanized hA2-27D-L1 expression vector was constructed inthe same way as in Example 8)-2-1. The obtained expression vector wasdesignated as “pCMA/hA2-27D-L1”.

8)-4-2 Construction of Humanized hA2-27D-L2 Type Light Chain ExpressionVector

A DNA fragment containing a sequence encoding humanized hA2-27D-L2, asshown in SEQ ID NO: 51, was synthesized (GeneArt Gene SynthesisService). A humanized hA2-27D-L2 expression vector was constructed inthe same way as in Example 8)-2-1. The obtained expression vector wasdesignated as “pCMA/hA2-27D-L2”.

8)-4-3 Construction of Humanized hA2-27D-L3 Type Light Chain ExpressionVector

A DNA fragment containing a sequence encoding humanized hA2-27D-L3, asshown in SEQ ID NO: 53, was synthesized (GeneArt Gene SynthesisService). A humanized hA2-27D-L3 expression vector was constructed inthe same way as in Example 8)-2-1. The obtained expression vector wasdesignated as “pCMA/hA2-27D-L3”.

8)-4-4 Construction of Humanized hA2-27D-L4 Type Light Chain ExpressionVector

A DNA fragment containing a sequence encoding humanized hA2-27D-L4, asshown in SEQ ID NO: 55, was synthesized (GeneArt Gene SynthesisService). A humanized hA2-27D-L4 expression vector was constructed inthe same way as in Example 8)-2-1. The obtained expression vector wasdesignated as “pCMA/hA2-27D-L4”.

8)-4-5 Construction of Humanized hA2-27D-L5 Type Light Chain ExpressionVector

A DNA fragment containing a sequence encoding humanized hA2-27D-L5, asshown in SEQ ID NO: 57, was synthesized (GeneArt Gene SynthesisService). A humanized hA2-27D-L5 expression vector was constructed inthe same way as in Example 8)-2-1. The obtained expression vector wasdesignated as “pCMA/hA2-27D-L5”.

8)-5 Preparation of Humanized A2-15A Antibody (IgG1) and HumanizedA2-27D Antibody (IgG1)

8)-5-1 Production of Humanized A2-15A Antibody (IgG1) and HumanizedA2-27D Antibody (IgG1)

Each antibody was obtained in the same way as in Example 5)-7.Specifically, humanized hA2-15A-H4/L6 was obtained by the combination ofpCMA-G1/hA2-15A-H4 constructed in Example 8)-1-2 and pCMA/hA2-15A-L6constructed in Example 8)-2-3. Humanized hA2-27D-H2/L2 was obtained bythe combination of pCMA-G1/hA2-27D-H2 constructed in Example 8)-3-2 andpCMA/hA2-27D-L2 constructed in Example 8)-4-2. Humanized hA2-27D-H3/L4was obtained by the combination of pCMA-G1/hA2-27D-H3 constructed inExample 8)-3-3 and pCMA/hA2-27D-L4 constructed in Example 8)-4-4.

8)-5-2 Two-Step Purification of Humanized A2-15A Antibody (IgG1) andHumanized A2-27D Antibody (IgG)

Each culture supernatant obtained in Example 8)-5-1 was purified in thesame way as in Example 5)-8.

Example 9 In Vitro Activity Evaluation of Humanized A2-15A Antibody(IgG1) and Humanized A2-27D Antibody (IgG1)

9)-1 Antibody Evaluation by Luciferase Reporter Assay

Humanized hA2-15A-H4/L6, humanized hA2-27D-H2/L2, and humanizedhA2-27D-H3/L4 prepared in Example 8)-5 were analyzed for theirinhibitory activity against ALK2-mediated intracellular signals using aluciferase reporter specific for BMP. HEPG2 cells were transfected withpGL4.26/Id1WT4F-luc (Genes Cells, 7, 949 (2002)) in the same way as inExample 6)-1. After 3 hours, the medium was replaced with freshFreeStyle 293 expression medium (Invitrogen Corp.). Then, each humanizedantibody and 10 ng/mL BMP7 (manufactured by Miltenyi Biotec) were addedthereto. On the next day, luciferase activity was measured.

The results are shown in FIG. 31. Humanized hA2-15A-H4/L6, humanizedhA2-27D-H2/L2, and humanized hA2-27D-H3/L4 were confirmed to inhibit, ina dose-dependent manner, BMP-specific luciferase activity induced byBMP7.

Example 10 Evaluation of Binding Activity of Humanized A2-15A Antibody(IgG1) and Humanized A2-27D Antibody (IgG1) Against Human ALK2

The dissociation constants of humanized hA2-15A-H4/L6, humanizedhA2-27D-H2/L2, and humanized hA2-27D-H3/L4 prepared in Example 8)-5 forthe antigen (Recombinant Human ALK2 Fc Chimera, Sino Biological Inc.)were measured in Biacore T200 (GE Healthcare Bio-Sciences Corp.) byusing the antigen immobilized as a ligand and each antibody as ananalyte. The antigen was added at 1.25 μg/mL for 60 seconds andimmobilized onto a sensor chip CM5 (GE Healthcare Bio-Sciences Corp.).The running buffer used was HBS-EP+ (10 mM HEPES (pH 7.4), 0.15 M NaCl,3 mM EDTA, and 0.05% Surfactant P20). Serially diluted solutions (0.2 to50 nM) of the antibody were added onto the antigen-immobilized chip at aflow rate of 30 μl/min for 300 seconds. Subsequently, the dissociationphase was monitored for 1800 seconds. A 10 mM glycine/hydrochloric acidsolution (pH 1.5) was added thereto as a regenerating solution at a flowrate of 10 μl/min for 30 seconds twice. The data was analyzed usingbivalent binding models of analytical software (BIAevaluation software,version 1.0) to calculate an association rate constant ka, adissociation rate constant kd, and a dissociation constant (KD;KD=kd/ka).

The results are shown in Table 1.

TABLE 1 Dissociation constants of humanized A2-15A antibody andhumanized A2-27D antibody Name KD(nM) 1 hA2-27D-H2/L2 5.7 2hA2-27D-H3/L4 4.3 3 hA2-15A-H4/L6 4.3

Example 11 Preparation of Humanized A2-15A Antibody (IgG2)

11)-1 Construction of Humanized IgG2 Type Heavy Chain Expression VectorpCMA-G2

pCMA-LK constructed in Example 5)-1 was digested with XbaI and PmeI. Theobtained DNA fragment except for the DNA sequence encoding the K chainsecretory signal and the human κ chain constant region was ligated witha DNA fragment (SEQ ID NO: 103) containing a nucleotide sequenceencoding the amino acid sequence of a human heavy chain secretory signalsequence and a human IgG2 constant region using In-Fusion Advantage PCRcloning kit (Clontech Laboratories, Inc.) to construct a chimeric andhumanized IgG2 type heavy chain expression vector pCMA-G2 having asignal sequence, a cloning site, and the nucleotide sequence encodingthe human IgG2 heavy chain constant region, downstream of the CMVpromoter.

11)-2 Construction of Humanized hA2-15A-H4 IgG2 Type Heavy ChainExpression Vector

A DNA fragment containing heavy chain variable region-encoding cDNA wasamplified using pCMA-G1/hA2-15A-H4 constructed in Example 8)-1-2 as atemplate, KOD-Plus-(Toyobo Co., Ltd.), and a primer set given below, andinserted at the restriction enzyme BlpI-cleaved site of the humanizedIgG2 type heavy chain expression vector pCMA-G2 using In-Fusion HD PCRcloning kit (Clontech Laboratories, Inc.) to construct a humanizedhA2-15A-H4 IgG2 type expression vector. The obtained expression vectorwas designated as “pCMA-G2/hA2-15A-H4”.

The nucleotide sequence of the humanized hA2-15A-H4 IgG2 type heavychain is shown in SEQ ID NO: 104, and the amino acid sequence thereof isshown in SEQ ID NO: 105. A sequence consisting of nucleotide numbers 1to 57 of the nucleotide sequence of SEQ ID NO: 104, a sequenceconsisting of nucleotide numbers 58 to 426 thereof, and a sequenceconsisting of nucleotide numbers 427 to 1404 thereof encode the signalsequence, the heavy chain variable region sequence, and the heavy chainconstant region sequence, respectively. A sequence consisting of aminoacid numbers 1 to 19 of the amino acid sequence of SEQ ID NO: 105, asequence consisting of amino acid numbers 20 to 142 thereof, and asequence consisting of amino acid numbers 143 to 468 thereof correspondto the signal sequence, the heavy chain variable region, and the heavychain constant region, respectively. The nucleotide sequence of SEQ IDNO: 104 and the amino acid sequence of SEQ ID NO: 105 are also describedin FIG. 32.

Primer set for humanized hA2-15A-H4 IgG2 type heavy chain(15A-H4-F; SEQ ID NO: 130)5′-CAGATGGGTGCTGAGCGAAGTGCAGCTGGTGGAATCTGGC-3′(15A-H4-R; SEQ ID NO: 131) 5′-CTTGGTGCTGGCTGAGCTGACGGTCACGAGGGTGCC-3′

11)-3 Production of Humanized A2-15A Antibody (IgG2)

The antibody was produced in the same way as in Example 5)-7. Ahumanized A2-15A antibody obtained by the combination ofpCMA-G2/hA2-15A-H4 constructed in Example 11)-2 and pCMA/hA2-15A-L6constructed Example 8)-2-3 was designated as “humanized hA2-15A-H4/L6(IgG2)”.

11)-4 Two-Step Purification of Humanized hA2-15A-H4/L6 (IgG2)

The culture supernatant obtained in Example 11)-3 was purified in thesame way as in Example 5)-8.

Example 12 Preparation of Humanized A2-27D Antibody (IgG1 LALA)

12)-1 Construction of Humanized hA2-27D-H2-LALA Type Heavy ChainExpression Vector

Mutations were introduced using pCMA-G1/hA2-27D-H2 constructed inExample 8)-3-2 as a template, a primer set given below, andKOD-Plus-Mutagenesis Kit (Toyobo Co., Ltd.) to construct ahA2-27D-H2-LALA type heavy chain expression vector. The constructedexpression vector was designated as “pCMA-G1/hA2-27D-H2-LALA”.

The nucleotide sequence of the humanized hA2-27D-H2-LALA type heavychain is shown in SEQ ID NO: 106, and the amino acid sequence thereof isshown in SEQ ID NO: 107. A sequence consisting of nucleotide numbers 1to 57 of the nucleotide sequence of SEQ ID NO: 106, a sequenceconsisting of nucleotide numbers 58 to 420 thereof, and a sequenceconsisting of nucleotide numbers 421 to 1410 thereof encode the signalsequence, the heavy chain variable region sequence, and the heavy chainconstant region sequence, respectively. A sequence consisting of aminoacid numbers 1 to 19 of the amino acid sequence of SEQ ID NO: 107, asequence consisting of amino acid numbers 20 to 140 thereof, and asequence consisting of amino acid numbers 141 to 470 thereof correspondto the signal sequence, the heavy chain variable region, and the heavychain constant region, respectively. The nucleotide sequence of SEQ IDNO: 106 and the amino acid sequence of SEQ ID NO: 107 are also describedin FIG. 33.

Primer set: (LALA-F; SEQ ID NO: 132)5′-GCGGGGGGACCCTCAGTCTTCCTCTTCCCC-3′ (LALA-R; SEQ ID NO: 133)5′-GGCTTCAGGTGCTGGGCAGGGTGGGCATGTG-3′

12)-2 Construction of Humanized hA2-27D-H3-LALA Type Heavy ChainExpression Vector

A hA2-27D-H3-LALA type heavy chain expression vector was constructed inthe same way as in Example 12)-1 using pCMA-G1/hA2-27D-H3 constructed inExample 8)-3-3 as a template. The constructed expression vector wasdesignated as “pCMA-G1/hA2-27D-H3-LALA”.

The nucleotide sequence of the humanized hA2-27D-H3-LALA type heavychain is shown in SEQ ID NO: 108, and the amino acid sequence thereof isshown in SEQ ID NO: 109. A sequence consisting of nucleotide numbers 1to 57 of the nucleotide sequence of SEQ ID NO: 108, a sequenceconsisting of nucleotide numbers 58 to 420 thereof, and a sequenceconsisting of nucleotide numbers 421 to 1410 thereof encode the signalsequence, the heavy chain variable region sequence, and the heavy chainconstant region sequence, respectively. A sequence consisting of aminoacid numbers 1 to 19 of the amino acid sequence of SEQ ID NO: 109, asequence consisting of amino acid numbers 20 to 140 thereof, and asequence consisting of amino acid numbers 141 to 470 thereof correspondto the signal sequence, the heavy chain variable region, and the heavychain constant region, respectively. The nucleotide sequence of SEQ IDNO: 108 and the amino acid sequence of SEQ ID NO: 109 are also describedin FIG. 34.

12)-3 Production of Humanized A2-27D Antibody (IgG1 LALA)

Each antibody was produced in the same way as in Example 5)-7. Ahumanized A2-27D antibody obtained by the combination ofpCMA-G1/hA2-27D-H2-LALA constructed in Example 12)-1 and pCMA/hA2-27D-L2constructed in Example 8)-4-2 was designated as “humanized hA2-27D-H2/L2(IgG1 LALA)”. A humanized A2-27D antibody obtained by the combination ofpCMA-G1/hA2-27D-H3-LALA constructed in Example 12)-2 and pCMA/hA2-27D-L4constructed in Example 8)-4-4 was designated as “humanized hA2-27D-H3/L4(IgG1 LALA)”.

12)-4 Two-Step Purification of Humanized A2-27D Antibody (IgG1 LALA)

Each culture supernatant obtained in Example 12)-3 was purified in thesame way as in Example 5)-8.

Example 13 In Vitro Activity Evaluation of Humanized A2-15A Antibody(IgG2) and Humanized A2-27D Antibody (IgG1 LALA)

13)-1 Antibody evaluation by luciferase reporter assay HumanizedA2-15A-H4/L6 (IgG2) prepared in Example 11)-4 and humanized A2-27D-H2/L2(IgG1 LALA) obtained in Example 12)-4 were analyzed for their inhibitoryactivity against ALK2-mediated intracellular signals using a luciferasereporter specific for BMP. HEPG2 cells were transfected withpGL4.26/Id1WT4F-luc (Genes Cells, 7, 949 (2002)) in the same way as inExample 6)-1. After 3 hours, the medium was replaced with freshFreeStyle 293 expression medium (Invitrogen Corp.). Then, each humanizedantibody and 2.5 ng/mL BMP9 were added thereto. On the next day,luciferase activity was measured in the same way as in Example 6)-1.

The results are shown in FIG. 35. Humanized A2-15A-H4/L6 (IgG2) andhumanized A2-27D-H2/L2 (IgG1 LALA) were confirmed to inhibit, in adose-dependent manner, BMP-specific luciferase activity induced by BMP9.

Example 14 Evaluation of Binding Activity of Humanized A2-15A Antibody(IgG2) and Humanized A2-27D Antibody (IgG1 LALA) Against Human ALK2

14)-1 Expression and Purification of Human ALK2 Extracellular Domain

DNA encoding a human ALK2 extracellular domain (polypeptide consistingof amino acid numbers 21 to 123 of the amino acid sequence of AccessionNo. NP_001096 of the NCBI protein database) was inserted into a vectorpET-28b(+) (Novagen/Merck KGaA, Catalog No: 69865) to construct aplasmid for the expression of a protein C-terminally linked to a His tagsequence. E. coli SHuffle T7 (New England Biolabs Inc., Catalog No:C3029H) was transformed with this plasmid and cultured in TB medium(Sigma-Aldrich Co. LLC, Catalog No: T0918). The E. coli thus culturedwas ultrasonicated, and the obtained bacterial cells were centrifuged.The supernatant was purified using HisTrap FF crude column (GEHealthcare Bio-Sciences Corp., Catalog No: 17-5286-01). Then, the humanALK2 extracellular domain was purified by electrophoresis using HiLoad26/600 Superdex 200 column (GE Healthcare Bio-Sciences Corp., CatalogNo: 28-9893-36) until a single band with a molecular weight of 12 kDawas obtained.

14)-2 Measurement of Ability of Humanized A2-15A Antibody (IgG2) andHumanized A2-27D Antibody (IgG1 LALA) to Bind to Antigen

The dissociation constants of humanized hA2-15A-H4/L6 (IgG2) prepared inExample 11)-4, and humanized hA2-27D-H2/L2 (IgG1 LALA) and humanizedhA2-27D-H3/L4 (IgG1 LALA) prepared in Example 12)-4 for the antigen(human ALK2 extracellular domain prepared in Example 14)-1) weremeasured using Biacore T200 (GE Healthcare Bio-Sciences Corp., Japan) bythe capture method which involves capturing each antibody as a ligandonto an immobilized anti-human IgG (Fc) antibody and assaying theantigen as an analyte. Approximately 1000 RU of the anti-human IgG (Fc)antibody (Human Antibody Capture kit, GE Healthcare Bio-Sciences Corp.)was covalently bound to a sensor chip CM5 (GE Healthcare Bio-SciencesCorp.) by the amine coupling method. Similarly, this antibody wasimmobilized onto a reference cell. The running buffer used wasHBS-EP+(10 mM HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.05%Surfactant P20). The antibody was added onto the anti-human IgG (Fc)antibody-immobilized chip for approximately 1 minute. Then, seriallydiluted solutions (0.78 to 200 nM) of the antigen were added thereto ata flow rate of 30 μl/min for 300 seconds. Subsequently, the dissociationphase was monitored for 600 seconds. A 3 M magnesium chloride solutionwas added thereto as a regenerating solution at a flow rate of 10 l/minfor 30 seconds. The data was analyzed using 1:1 binding models ofanalytical software (BIAevaluation software, version 1.0) to calculatean association rate constant ka, a dissociation rate constant kd, and adissociation constant (KD; KD=kd/ka).

The results are shown in Table 2.

TABLE 2 Dissociation constants of humanized A2-15A antibody (IgG2) andhumanized A2-27D antibody (IgG1 LALA) Name KD(nM) 1 hA2-15A-H4/L6, IgG217.4 2 hA2-27D-H2/L2, IgG1 LALA 13.6 3 hA2-27D-H3/L4, IgG1 LALA 13.3

Example 15 Evaluation of In Vivo Activity of Humanized A2-15A Antibody(IgG2) and Humanized A2-27D Antibody (IgG1 LALA)

Humanized hA2-15A-H4/L6 (IgG2) prepared in Example 11)-4 and humanizedhA2-27D-H2/L2 (IgG1 LALA) prepared in Example 12)-4 were analyzed fortheir inhibitory activity against ectopic ossification using mousemodels with ectopic ossification caused by the transplantation of BMP7.CollaTape (manufactured by Zimmer Biomet Dental K.K.) cut into a roundshape of 4 mm in diameter was impregnated with 2.5 gig of BMP7(manufactured by Miltenyi Biotec), frozen overnight −80° C., and thendried in vacuum. Skin hair near the thigh bone of each mouse (C57BL/6;8-9 weeks old) was shaved, and an incision was made in this area underanesthesia by the aspiration of isoflurane. The freeze-dried filterpaper was transplanted into skeletal muscle. Two weeks after thetransplantation, calcified ectopic bone was analyzed as ectopicossification by micro-CT (manufactured by Comscantecno Co., Ltd.). Then,the ectopic bone in the skeletal muscle was isolated, and the weight wasmeasured. Each antibody was subcutaneously administered once a week(days −1 and 6 when the transplantation day was defined as day 0). Theantibody was diluted with a solvent (HBSor) such that its concentrationwas 1, 3, or 10 mg/kg. Control human IgG (manufactured by JacksonImmunoResearch Laboratories, Inc.) was adjusted to 10 mg/kg with asolvent and administered to mice in a control group.

The results are shown in FIG. 36. Humanized hA2-15A-H4/L6 (IgG2) andhumanized hA2-27D-H2/L2 (IgG1 LALA) were confirmed to suppressBMP7-induced ectopic osteoinduction in mouse skeletal muscle tissues.

Example 16 Epitope Analysis of A2-27D Antibody

16)-1 Preparation of Human Chimeric cA2-27D Fab Fragment

A Fab fragment was prepared from the human chimeric cA2-27D antibodyobtained in Example 5)-8 using Pierce Fab Preparation Kit.

16)-2 Crystallization and Structural Analysis of Human Chimeric cA2-27DFab Fragment and ALK2-ECD Complex

The protein complex of the human chimeric cA2-27D Fab fragment obtainedin Example 16)-1 and the ALK2-ECD prepared according to Example 14 wereconcentrated to 2.4 mg/mL and used in the crystallization trialemploying vapor diffusion method. To 0.5 μL of the protein solution, anequal amount of a precipitant solution (2% (v/v) Tacsimate (pH 7.0), 100mM HEPES (pH 7.5), and 20% (w/v) polyethylene glycol 3,350) was added,and the resulting solution was placed in a sealed container containing50 μL of a precipitant solution such that these solutions had no contactwith each other. The container was left standing at 20° C. Three dayslater, 0.1 mm×0.1 mm×0.1 mm single crystals were obtained. The obtainedcrystals were dipped in a precipitant solution supplemented with 20%(v/v) glycerol, and subsequently frozen in liquid nitrogen. X-raydiffraction data was collected under 95 K nitrogen stream using BLA ofKEK Photon Factory. Diffraction intensity was digitized from theobtained diffraction image using software XDS (Acta Cryst. (2010). D66,125-132) to determine crystal structure factors. The crystals were inthe body-centered monoclinic crystal system with a space group C121 andunit cells of a=c=119.39 angstroms, b=37.32 angstroms, and β=92.54. Themolecular replacement method was performed using the obtained structurefactors and the three-dimensional structure coordinates of Fab (antibodystructure determined by the past crystal structure analysis was used) todetermine the phases. Software phaser (CCP4: Collaborative ComputationalProject No. 4) was used in calculation. The crystal contained onecomplex in the asymmetric unit. Structure refinement was performed usingsoftware Refmac5 (CCP4), and model correction was performed usingsoftware coot. This operation was repetitively performed to obtain afinal R factor of 22.3% and a free R factor of 26.7% with a resolutionof 2.6 angstroms. The model consists of one complex and contains aminoacid numbers 1 to 211 of the A2-27D Fab L chain, amino acid numbers 1 to134 and 141 to 223 of the A2-27D Fab H chain, amino acid numbers 11 to89 of ALK2-ECD, and 61 water molecules. The determined amino acidresidues of ALK2-ECD located within 4 angstroms from A2-27D Fab are asfollows: Glu18, Gly19, Ile39, Asn40, Asp41, Gly42, Phe43, His44, Val145,Tyr46, Asn82, Thr84, Gln86, and Leu87. The ribbon model of the wholecomplex is shown in FIG. 37.

Example 17 Design of Humanized A2-11E Antibody and Humanized A2-25CAntibody

17)-1 Design of Humanized hA2-11E

17)-1-1 Molecular Modeling of A2-11E Variable Regions

The molecular modeling of the A2-11E variable regions was carried out bya method generally known as homology modeling (Methods in Enzymology,203, 121-153, (1991)). The variable regions of A2-11E determined abovewere compared with the primary sequences (three-dimensional structuresderived from X-ray crystal structures are available) of humanimmunoglobulin variable regions registered in Protein Data Bank (Nuc.Acid Res. 35, D301-D303 (2007)). As a result, 3BN9 was selected becauseof having the highest sequence identity to the heavy and light chainvariable regions of A2-11E. The three-dimensional structures offramework regions were prepared as a “framework model” by combining thecoordinates of 3BN9. Subsequently, the typical conformation of each CDRwas incorporated into the framework model.

Finally, energy minimization calculation for excluding disadvantageousinteratomic contact was conducted in order to obtain possible molecularmodels of the A2-11E variable regions in terms of energy. Theseprocedures were performed using a commercially available proteinthree-dimensional structure analysis program Discovery Studio(manufactured by Accelrys).

17)-1-2 Design of Amino Acid Sequence of Humanized hA2-11E

Humanized hA2-11E was constructed by a method generally known as CDRgrafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Anacceptor antibody was selected on the basis of the identity of aminoacids in framework regions.

The sequences of the framework regions of A2-11E were compared with theframework regions of human subgroup consensus sequences. As a result, ahuman γ chain subgroup 3 consensus sequence and a human κ chain subgroup1 consensus sequence specified by KABAT et al. (Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service NationalInstitutes of Health, Bethesda, Md. (1991)) were selected as acceptorsdue to their high sequence identity as to framework regions. The aminoacid residues of the framework regions of the human γ chain subgroup 3consensus sequence and the human κ chain subgroup 1 consensus sequencewere aligned with the amino acid residues of the A2-11E frameworkregions to identify the numbers of amino acids that did not matchtherebetween. The numbers of these residues were analyzed using thethree-dimensional model of A2-11E constructed above. Then, the donorresidues to be grafted onto the acceptors were selected according to thecriteria provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86,10029-10033 (1989)). Some donor residues thus selected were transferredto the acceptor antibody to construct the humanized hA2-11E sequence asdescribed in Examples below.

17)-2 Humanization of A2-11E Heavy Chain

17)-2-1 Humanized hA2-11E-H3 Type Heavy Chain

A humanized hA2-11E heavy chain designed from the A2-11E heavy chain ofSEQ ID NO: 2 of the Sequence Listing by the replacement of amino acidposition 7 (threonine) with serine, amino acid position 16 (arginine)with glycine, amino acid position 19 (lysine) with arginine, amino acidposition 23 (valine) with alanine, amino acid position 48 (isoleucine)with valine, amino acid position 61 (proline) with alanine, amino acidposition 69 (alanine) with threonine, amino acid position 75 (alanine)with serine, amino acid position 76 (glutamic acid) with lysine, aminoacid position 88 (serine) with alanine, and amino acid position 93(threonine) with valine was designated as “hA2-11E-H3 type heavy chain”.

The amino acid sequence of the humanized hA2-11E-H3 type heavy chain isdescribed in SEQ ID NO: 111 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19 of the amino acid sequence ofSEQ ID NO: 111, a sequence consisting of amino acid numbers 20 to 137thereof, and a sequence consisting of amino acid numbers 138 to 467thereof correspond to the signal sequence, the heavy chain variableregion, and the heavy chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 111is described in SEQ ID NO: 110 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 57 of the nucleotide sequence ofSEQ ID NO: 110, a sequence consisting of nucleotide numbers 58 to 411thereof, and a sequence consisting of nucleotide numbers 412 to 1401thereof encode the signal sequence, the heavy chain variable regionsequence, and the heavy chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 110 and the amino acid sequence ofSEQ ID NO: 111 are also described in FIG. 38.

17)-2-2 Humanized hA2-11E-H4 Type Heavy Chain

A humanized hA2-11E heavy chain designed from the A2-11E heavy chain ofSEQ ID NO: 2 of the Sequence Listing by the replacement of amino acidposition 7 (threonine) with serine, amino acid position 16 (arginine)with glycine, amino acid position 19 (lysine) with arginine, amino acidposition 23 (valine) with alanine, amino acid position 69 (alanine) withthreonine, amino acid position 75 (alanine) with serine, amino acidposition 76 (glutamic acid) with lysine, amino acid position 88 (serine)with alanine, and amino acid position 93 (threonine) with valine wasdesignated as “hA2-11E-H4 type heavy chain”.

The amino acid sequence of the humanized hA2-11E-H4 type heavy chain isdescribed in SEQ ID NO: 113 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19 of the amino acid sequence ofSEQ ID NO: 113, a sequence consisting of amino acid numbers 20 to 137thereof, and a sequence consisting of amino acid numbers 138 to 467thereof correspond to the signal sequence, the heavy chain variableregion, and the heavy chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 113is described in SEQ ID NO: 112 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 57 of the nucleotide sequence ofSEQ ID NO: 112, a sequence consisting of nucleotide numbers 58 to 411thereof, and a sequence consisting of nucleotide numbers 412 to 1401thereof encode the signal sequence, the heavy chain variable regionsequence, and the heavy chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 112 and the amino acid sequence ofSEQ ID NO: 113 are also described in FIG. 39.

17)-3 Humanization of A2-11E Light Chain

17)-3-1 Humanized hA2-11E-L2 Type Light Chain

A humanized hA2-11E light chain designed from the A2-11E light chain ofSEQ ID NO: 4 of the Sequence Listing by the replacement of amino acidnumber 10 (leucine) with serine, amino acid position 21 (leucine) withisoleucine, amino acid number 22 (serine) with threonine, amino acidnumber 40 (leucine) with proline, amino acid number 42 (glutamic acid)with lysine, amino acid number 58 (isoleucine) with valine, amino acidnumber 83 (valine) with phenylalanine, amino acid number 85 (isoleucine)with threonine, amino acid number 87 (phenylalanine) with tyrosine,amino acid number 99 (proline) with glutamine, amino acid number 103(leucine) with valine, amino acid number 105 (leucine) with isoleucine,and amino acid number 108 (alanine) with threonine was designated as“hA2-11E-L2 type light chain”.

The amino acid sequence of the humanized hA2-11E-L2 type light chain isdescribed in SEQ ID NO: 115 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20 of the amino acid sequence ofSEQ ID NO: 115, a sequence consisting of amino acid numbers 21 to 128thereof, and a sequence consisting of amino acid numbers 129 to 233thereof correspond to the signal sequence, the light chain variableregion, and the light chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 115is described in SEQ ID NO: 114 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 60 of the nucleotide sequence ofSEQ ID NO: 114, a sequence consisting of nucleotide numbers 61 to 384thereof, and a sequence consisting of nucleotide numbers 385 to 699thereof encode the signal sequence, the light chain variable regionsequence, and the light chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 114 and the amino acid sequence ofSEQ ID NO: 115 are also described in FIG. 40.

17)-3-2 Humanized hA2-11E-L3 Type Light Chain

A humanized hA2-11E light chain designed from the A2-11E light chain ofSEQ ID NO: 4 of the Sequence Listing by the replacement of amino acidnumber 10 (leucine) with serine, amino acid number 21 (leucine) withisoleucine, amino acid number 22 (serine) with threonine, amino acidnumber 40 (leucine) with proline, amino acid number 42 (glutamic acid)with lysine, amino acid number 83 (valine) with phenylalanine, aminoacid number 85 (isoleucine) with threonine, amino acid number 87(phenylalanine) with tyrosine, amino acid number 99 (proline) withglutamine, amino acid number 103 (leucine) with valine, amino acidnumber 105 (leucine) with isoleucine, and amino acid number 108(alanine) with threonine was designated as “hA2-11E-L3 type lightchain”.

The amino acid sequence of the humanized hA2-11E-L3 type light chain isdescribed in SEQ ID NO: 117 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20 of the amino acid sequence ofSEQ ID NO: 117, a sequence consisting of amino acid numbers 21 to 128thereof, and a sequence consisting of amino acid numbers 129 to 233thereof correspond to the signal sequence, the light chain variableregion, and the light chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 117is described in SEQ ID NO: 116 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 60 of the nucleotide sequence ofSEQ ID NO: 116, a sequence consisting of nucleotide numbers 61 to 384thereof, and a sequence consisting of nucleotide numbers 385 to 699thereof encode the signal sequence, the light chain variable regionsequence, and the light chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 116 and the amino acid sequence ofSEQ ID NO: 117 are also described in FIG. 41.

17)-3-3 Humanized hA2-11E-L4 Type Light Chain

A humanized hA2-11E light chain designed from the A2-11E light chain ofSEQ ID NO: 4 of the Sequence Listing by the replacement of amino acidnumber 10 (leucine) with serine, amino acid number 21 (leucine) withisoleucine, amino acid number 40 (leucine) with proline, amino acidnumber 83 (valine) with phenylalanine, amino acid number 103 (leucine)with valine, amino acid number 105 (leucine) with isoleucine, and aminoacid number 108 (alanine) with threonine was designated as “hA2-11E-L4type light chain”.

The amino acid sequence of the humanized hA2-11E-L4 type light chain isdescribed in SEQ ID NO: 119 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20 of the amino acid sequence ofSEQ ID NO: 119, a sequence consisting of amino acid numbers 21 to 128thereof, and a sequence consisting of amino acid numbers 129 to 233thereof correspond to the signal sequence, the light chain variableregion, and the light chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 119is described in SEQ ID NO: 118 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 60 of the nucleotide sequence ofSEQ ID NO: 118, a sequence consisting of nucleotide numbers 61 to 384thereof, and a sequence consisting of nucleotide numbers 385 to 699thereof encode the signal sequence, the light chain variable regionsequence, and the light chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 118 and the amino acid sequence ofSEQ ID NO: 119 are also described in FIG. 42.

17)-4 Design of Humanized hA2-11E by Combination of Heavy Chain andLight Chain

An antibody consisting of the humanized hA2-11E-H3 type heavy chain andthe humanized hA2-11E-L2 type light chain was designed and designated as“humanized hA2-11E-H3/L2” (also referred to as “hA2-11E-H3/L2”). Anantibody consisting of the humanized hA2-11E-H3 type heavy chain and thehumanized hA2-11E-L3 type light chain was designed and designated as“humanized hA2-11E-H3/L3” (also referred to as “hA2-11E-H3/L3”). Anantibody consisting of the humanized hA2-11E-H3 type heavy chain and thehumanized hA2-11E-L4 type light chain was designed and designated as“humanized hA2-11E-H3/L4” (also referred to as “hA2-11E-H3/L4”). Anantibody consisting of the humanized hA2-11E-H4 type heavy chain and thehumanized hA2-11E-L2 type light chain was designed and designated as“humanized hA2-11E-H4/L2” (also referred to as “hA2-11E-H4/L2”). Anantibody consisting of the humanized hA2-11E-H4 type heavy chain and thehumanized hA2-11E-L3 type light chain was designed and designated as“humanized hA2-11E-H4/L3” (also referred to as “hA2-11E-H4/L3”). Anantibody consisting of the humanized hA2-11E-H4 type heavy chain and thehumanized hA2-11E-L4 type light chain was designed and designated as“humanized hA2-11E-H4/L4” (also referred to as “hA2-11E-H4/L4”). Thesedesigned antibodies can be prepared according to Example 18 andevaluated according to Examples 2 and 4.

17)-5 Design of Humanized hA2-25C

17)-5-1 Molecular Modeling of A2-25C Variable Regions

The molecular modeling of the A2-25C variable regions was carried out bya method generally known as homology modeling (Methods in Enzymology,203, 121-153, (1991)). The variable regions of AA2-25C determined abovewere compared with the primary sequences (three-dimensional structuresderived from X-ray crystal structures are available) of humanimmunoglobulin variable regions registered in Protein Data Bank (Nuc.Acid Res. 35, D301-D303 (2007)). As a result, 3BN9 was selected becauseof having the highest sequence identity to the heavy and light chainvariable regions of A2-25C. The three-dimensional structures offramework regions were prepared as a “framework model” by combining thecoordinates of 3BN9. Subsequently, the typical conformation of each CDRwas incorporated into the framework model.

Finally, energy minimization calculation for excluding disadvantageousinteratomic contact was conducted in order to obtain possible molecularmodels of the A2-25C variable regions in terms of energy. Theseprocedures were performed using a commercially available proteinthree-dimensional structure analysis program Discovery Studio(manufactured by Accelrys).

17)-5-2 Design of Amino Acid Sequence of Humanized hA2-25C

Humanized hA2-25C was constructed by a method generally known as CDRgrafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Anacceptor antibody was selected on the basis of the identity of aminoacids in framework regions.

The sequences of the framework regions of A2-25C were compared with theframework regions of human subgroup consensus sequences. As a result, ahuman γ chain subgroup 3 consensus sequence and a human κ chain subgroup1 consensus sequence specified by KABAT et al. (Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service NationalInstitutes of Health, Bethesda, Md. (1991)) were selected as acceptorsdue to their high sequence identity as to framework regions. The aminoacid residues of the framework regions of the human γ chain subgroup 3consensus sequence and the human κ chain subgroup 1 consensus sequencewere aligned with the amino acid residues of the A2-25C frameworkregions to identify the numbers of amino acids that did not matchtherebetween. The numbers of these residues were analyzed using thethree-dimensional model of A2-25C constructed above. Then, the donorresidues to be grafted onto the acceptors were selected according to thecriteria provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86,10029-10033 (1989)). Some donor residues thus selected were transferredto the acceptor antibody to construct the humanized hA2-25C sequence asdescribed in Examples below.

17)-6 Humanization of A2-25C Heavy Chain

17)-6-1 Humanized hA2-25C-H3 Type Heavy Chain

A humanized hA2-25C heavy chain designed from the A2-25C heavy chain ofSEQ ID NO: 10 of the Sequence Listing by the replacement of amino acidnumber 19 (lysine) with arginine, amino acid number 38 (cysteine) witharginine, amino acid number 42 (threonine) with glycine, amino acidnumber 63 (threonine) with serine, amino acid number 75 (alanine) withserine, amino acid number 84 (aspartic acid) with asparagine, amino acidnumber 88 (serine) with alanine, amino acid number 93 (threonine) withvaline, amino acid number 112 (valine) with threonine, and amino acidnumber 113 (methionine) with leucine was designated as “hA2-25C-H3 typeheavy chain”.

The amino acid sequence of the humanized hA2-25C-H3 type heavy chain isdescribed in SEQ ID NO: 121 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19 of the amino acid sequence ofSEQ ID NO: 121, a sequence consisting of amino acid numbers 20 to 137thereof, and a sequence consisting of amino acid numbers 138 to 467thereof correspond to the signal sequence, the heavy chain variableregion, and the heavy chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 121is described in SEQ ID NO: 120 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 57 of the nucleotide sequence ofSEQ ID NO: 120, a sequence consisting of nucleotide numbers 58 to 411thereof, and a sequence consisting of nucleotide numbers 412 to 1401thereof encode the signal sequence, the heavy chain variable regionsequence, and the heavy chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 120 and the amino acid sequence ofSEQ ID NO: 121 are also described in FIG. 43.

17)-6-2 Humanized hA2-25C-H4 Type Heavy Chain

A humanized hA2-25C heavy chain designed from the A2-25C heavy chain ofSEQ ID NO: 10 of the Sequence Listing by the replacement of amino acidnumber 19 (lysine) with arginine, amino acid number 38 (cysteine) witharginine, amino acid number 42 (threonine) with glycine, amino acidnumber 63 (threonine) with serine, amino acid number 75 (alanine) withserine, amino acid number 84 (aspartic acid) with asparagine, amino acidnumber 88 (serine) with alanine, and amino acid number 113 (methionine)with leucine was designated as “hA2-25C-H4 type heavy chain”.

The amino acid sequence of the humanized hA2-25C-H4 type heavy chain isdescribed in SEQ ID NO: 123 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 19 of the amino acid sequence ofSEQ ID NO: 123, a sequence consisting of amino acid numbers 20 to 137thereof, and a sequence consisting of amino acid numbers 138 to 467thereof correspond to the signal sequence, the heavy chain variableregion, and the heavy chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 123is described in SEQ ID NO: 122 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 57 of the nucleotide sequence ofSEQ ID NO: 122, a sequence consisting of nucleotide numbers 58 to 411thereof, and a sequence consisting of nucleotide numbers 412 to 1401thereof encode the signal sequence, the heavy chain variable regionsequence, and the heavy chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 122 and the amino acid sequence ofSEQ ID NO: 123 are also described in FIG. 44.

17)-7 Humanization of A2-25C Light Chain

17)-7-1 Humanized hA2-25C-L1 Type Light Chain

A humanized hA2-25C light chain designed from the A2-25C light chain ofSEQ ID NO: 12 of the Sequence Listing by the replacement of amino acidnumber 9 (alanine) with serine, amino acid number 15 (leucine) withvaline, amino acid number 16 (glutamic acid) with glycine, amino acidnumber 17 (glutamic acid) with aspartic acid, amino acid number 18(isoleucine) with arginine, amino acid number 43 (serine) with alanine,amino acid number 45 (glutamine) with lysine, amino acid number 66(arginine) with glycine, amino acid number 70 (glutamine) with asparticacid, amino acid number 71 (tyrosine) with phenylalanine, amino acidnumber 72 (serine) with threonine, amino acid number 74 (lysine) withthreonine, amino acid number 77 (arginine) with serine, amino acidnumber 79 (arginine) with glutamine, amino acid number 80 (valine) withproline, amino acid number 83 (isoleucine) with phenylalanine, aminoacid number 84 (glycine) with alanine, amino acid number 85 (isoleucine)with threonine, amino acid number 100 (serine) with glutamine, aminoacid number 104 (leucine) with valine, and amino acid number 109(alanine) with threonine was designated as “hA2-25C-L1 type lightchain”.

The amino acid sequence of the humanized hA2-25C-L1 type light chain isdescribed in SEQ ID NO: 125 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20 of the amino acid sequence ofSEQ ID NO: 125, a sequence consisting of amino acid numbers 21 to 129thereof, and a sequence consisting of amino acid numbers 130 to 234thereof correspond to the signal sequence, the light chain variableregion, and the light chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 125is described in SEQ ID NO: 124 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 60 of the nucleotide sequence ofSEQ ID NO: 124, a sequence consisting of nucleotide numbers 61 to 387thereof, and a sequence consisting of nucleotide numbers 388 to 702thereof encode the signal sequence, the light chain variable regionsequence, and the light chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 124 and the amino acid sequence ofSEQ ID NO: 125 are also described in FIG. 45.

17)-7-2 Humanized hA2-25C-L2 Type Light Chain

A humanized hA2-25C light chain designed from the A2-25C light chain ofSEQ ID NO: 12 of the Sequence Listing by the replacement of amino acidnumber 9 (alanine) with serine, amino acid number 15 (leucine) withvaline, amino acid number 16 (glutamic acid) with glycine, amino acidnumber 17 (glutamic acid) with aspartic acid, amino acid number 18(isoleucine) with arginine, amino acid number 43 (serine) with alanine,amino acid number 45 (glutamine) with lysine, amino acid number 70(glutamine) with aspartic acid, amino acid number 72 (serine) withthreonine, amino acid number 74 (lysine) with threonine, amino acidnumber 77 (arginine) with serine, amino acid number 79 (arginine) withglutamine, amino acid number 80 (valine) with proline, amino acid number83 (isoleucine) with phenylalanine, amino acid number 84 (glycine) withalanine, amino acid number 85 (isoleucine) with threonine, amino acidnumber 100 (serine) with glutamine, amino acid number 104 (leucine) withvaline, and amino acid number 109 (alanine) with threonine wasdesignated as “hA2-25C-L2 type light chain”.

The amino acid sequence of the humanized hA2-25C-L2 type light chain isdescribed in SEQ ID NO: 127 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20 of the amino acid sequence ofSEQ ID NO: 127, a sequence consisting of amino acid numbers 21 to 129thereof, and a sequence consisting of amino acid numbers 130 to 234thereof correspond to the signal sequence, the light chain variableregion, and the light chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 127is described in SEQ ID NO: 126 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 60 of the nucleotide sequence ofSEQ ID NO: 126, a sequence consisting of nucleotide numbers 61 to 387thereof, and a sequence consisting of nucleotide numbers 388 to 702thereof encode the signal sequence, the light chain variable regionsequence, and the light chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 126 and the amino acid sequence ofSEQ ID NO: 127 are also described in FIG. 46.

17)-7-3 Humanized hA2-25C-L3 Type Light Chain

A humanized hA2-25C light chain designed from the A2-25C light chain ofSEQ ID NO: 12 of the Sequence Listing by the replacement of amino acidnumber 9 (alanine) with serine, amino acid number 15 (leucine) withvaline, amino acid number 16 (glutamic acid) with glycine, amino acidnumber 17 (glutamic acid) with aspartic acid, amino acid number 18(isoleucine) with arginine, amino acid number 45 (glutamine) withlysine, amino acid number 72 (serine) with threonine, amino acid number74 (lysine) with threonine, amino acid number 77 (arginine) with serine,amino acid number 79 (arginine) with glutamine, amino acid number 80(valine) with proline, amino acid number 83 (isoleucine) withphenylalanine, amino acid number 84 (glycine) with alanine, amino acidnumber 85 (isoleucine) with threonine, amino acid number 104 (leucine)with valine, and amino acid number 109 (alanine) with threonine wasdesignated as “hA2-25C-L3 type light chain”.

The amino acid sequence of the humanized hA2-25C-L3 type light chain isdescribed in SEQ ID NO: 129 of the Sequence Listing. A sequenceconsisting of amino acid numbers 1 to 20 of the amino acid sequence ofSEQ ID NO: 129, a sequence consisting of amino acid numbers 21 to 129thereof, and a sequence consisting of amino acid numbers 130 to 234thereof correspond to the signal sequence, the light chain variableregion, and the light chain constant region, respectively. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 129is described in SEQ ID NO: 128 of the Sequence Listing. A sequenceconsisting of nucleotide numbers 1 to 60 of the nucleotide sequence ofSEQ ID NO: 128, a sequence consisting of nucleotide numbers 61 to 387thereof, and a sequence consisting of nucleotide numbers 388 to 702thereof encode the signal sequence, the light chain variable regionsequence, and the light chain constant region sequence, respectively.The nucleotide sequence of SEQ ID NO: 128 and the amino acid sequence ofSEQ ID NO: 129 are also described in FIG. 47.

17)-8 Design of Humanized hA2-25C by Combination of Heavy Chain andLight Chain

An antibody consisting of the humanized hA2-25C-H3 type heavy chain andthe humanized hA2-25C-L1 type light chain was designed and designated as“humanized hA2-25C-H3/L1 (also referred to as “hA2-25C-H3/L1”). Anantibody consisting of the humanized hA2-25C-H3 type heavy chain and thehumanized hA2-25C-L2 type light chain was designed and designated as“humanized hA2-25C-H3/L2” (also referred to as “hA2-25C-H3/L2”). Anantibody consisting of the humanized hA2-125C-H3 type heavy chain andthe humanized hA2-25C-L3 type light chain was designed and designated as“humanized hA2-25C-H3/L3” (also referred to as “hA2-25C-H3/L3”). Anantibody consisting of the humanized hA2-25C-H4 type heavy chain and thehumanized hA2-25C-L1 type light chain was designed and designated as“humanized hA2-25C-H4/L1” (also referred to as “hA2-25C-H4/L1”). Anantibody consisting of the humanized hA2-25C-H4 type heavy chain and thehumanized hA2-25C-L2 type light chain was designed and designated as“humanized hA2-25C-H4/L2” (also referred to as “hA2-25C-H4/L2”). Anantibody consisting of the humanized hA2-25C-H4 type heavy chain and thehumanized hA2-25C-L3 type light chain was designed and designated as“humanized hA2-25C-H4/L3” (also referred to as “hA2-25C-H4/L3”). Thesedesigned antibodies can be prepared according to Example 18 andevaluated according to Examples 2 and 4.

Example 18 Construction and Preparation of Vectors for Humanized A2-11EAntibody (IgG1) and Humanized A2-25C Antibody (IgG)

18)-1 Construction of Humanized A2-11E Heavy Chain Expression Vector

18)-1-1 Construction of Humanized hA2-11E-H3 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-11E-H3 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 428of the nucleotide sequence of humanized hA2-11E-H3 of SEQ ID NO: 110 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-11E-H3 expression vector was constructed in the same way as inExample 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-11E-H3”.

18)-1-2 Construction of Humanized hA2-11E-H4 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-11E-H4 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 428of the nucleotide sequence of humanized hA2-11E-H4 of SEQ ID NO: 112 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-11E-H4 expression vector was constructed in the same way as inExample 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-11E-H4”.

18)-2 Construction of Humanized A2-11E Light Chain Expression Vector

18)-2-1 Construction of Humanized hA2-11E-L2 Type Light Chain ExpressionVector

A DNA fragment containing a humanized hA2-11E-L2 variableregion-encoding DNA sequence represented by nucleotide numbers 37 to 399of the nucleotide sequence of humanized hA2-11E-L2 of SEQ ID NO: 114 wassynthesized (GeneArt Artificial Gene Synthesis Service). The DNAfragment containing a humanized hA2-11E-L2 variable region-encoding DNAsequence was amplified using the synthesized DNA fragment as a template,KOD-Plus- (Toyobo Co., Ltd.), and a primer set given below, and insertedat the restriction enzyme BsiWI-cleaved site of the chimeric andhumanized antibody light chain expression vector pCMA-LK constructed inExample 5)-1 using In-Fusion HD PCR cloning kit (Clontech Laboratories,Inc.) to construct a humanized hA2-11E-L2 expression vector. Theobtained expression vector was designated as “pCMA/hA2-11E-L2”.

Primer set (CM-LKF; SEQ ID NO: 134) 5′-CTGTGGATCTCCGGCGCGTACGGC-3′(KCL-Inf-R; SEQ ID NO: 135) 5′-GGAGGGGGCGGCCACCGTACG-3′

18)-2-2 Construction of Humanized hA2-11E-L3 Type Light Chain ExpressionVector

A DNA fragment containing a humanized hA2-11E-L3 variableregion-encoding DNA sequence represented by nucleotide numbers 37 to 399of the nucleotide sequence of humanized hA2-11E-L3 of SEQ ID NO: 116 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-11E-L3 expression vector was constructed in the same way as inExample 18)-2-1. The obtained expression vector was designated as“pCMA/hA2-11E-L3”.

18)-2-3 Construction of Humanized hA2-11E-L4 Type Light Chain ExpressionVector

A DNA fragment containing a humanized hA2-11E-L4 variableregion-encoding DNA sequence represented by nucleotide numbers 37 to 339of the nucleotide sequence of humanized hA2-11E-L4 of SEQ ID NO: 118 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-11E-L4 expression vector was constructed in the same way as inExample 18)-2-1. The obtained expression vector was designated as“pCMA/hA2-11E-L4”.

18)-3 Construction of Humanized A2-25C Heavy Chain Expression Vector

18)-3-1 Construction of Humanized hA2-25C-H3 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-25C-H3 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 428of the nucleotide sequence of humanized hA2-25C-H3 of SEQ ID NO: 120 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-25C-H3 expression vector was constructed in the same way as inExample 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-25C-H3”.

18)-3-2 Construction of Humanized hA2-25C-H4 Type Heavy Chain ExpressionVector

A DNA fragment containing a humanized hA2-25C-H4 variableregion-encoding DNA sequence represented by nucleotide numbers 36 to 428of the nucleotide sequence of humanized hA2-25C-H4 of SEQ ID NO: 122 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-25C-H4 expression vector was constructed in the same way as inExample 8)-1-1. The obtained expression vector was designated as“pCMA-G1/hA2-25C-H4”.

18)-4 Construction of Humanized A2-25C Light Chain Expression Vector

18)-4-1 Construction of Humanized hA2-25C-L1 Type Light Chain ExpressionVector

A DNA fragment containing a humanized hA2-25C-L1 variableregion-encoding DNA sequence represented by nucleotide numbers 37 to 402of the nucleotide sequence of humanized hA2-25C-L1 of SEQ ID NO: 124 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-25C-L1 expression vector was constructed in the same way as inExample 18)-2-1. The obtained expression vector was designated as“pCMA/hA2-25C-L 1”.

18)-4-2 Construction of Humanized hA2-25C-L2 Type Light Chain ExpressionVector

A DNA fragment containing a humanized hA2-25C-L2 variableregion-encoding DNA sequence represented by nucleotide numbers 37 to 402of the nucleotide sequence of humanized hA2-25C-L2 of SEQ ID NO: 126 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-25C-L2 expression vector was constructed in the same way as inExample 18)-2-1. The obtained expression vector was designated as“pCMA/hA2-25C-L2”.

18)-4-3 Construction of Humanized hA2-25C-L3 Type Light Chain ExpressionVector

A DNA fragment containing a humanized hA2-25C-L3 variableregion-encoding DNA sequence represented by nucleotide numbers 37 to 402of the nucleotide sequence of humanized hA2-25C-L3 of SEQ ID NO: 128 wassynthesized (GeneArt Artificial Gene Synthesis Service). A humanizedhA2-25C-L3 expression vector was constructed in the same way as inExample 18)-2-1. The obtained expression vector was designated as“pCMA/hA2-25C-L3”.

18)-5 Small-Scale Production of Humanized A2-11E Antibody (IgG1) andHumanized A2-25C Antibody (IgG)

FreeStyle 293F cells (Invitrogen Corp.) were subcultured and culturedaccording to the manual.

1×10⁷ FreeStyle 293F cells (Invitrogen Corp.) in the logarithmic growthphase were diluted to 9.6 mL with FreeStyle 293 expression medium(Invitrogen Corp.), then inoculated to 30 mL Square Storage Bottle(Nalgene/Thermo Fisher Scientific Inc.), and shake-cultured at 90 rpm at37° C. for 1 hour in an 8% C02 incubator. 30 μg of polyethyleneimine(Polysciences #24765) was dissolved in 200 μL of Opti-Pro SFM(Invitrogen Corp.). Next, each light chain expression vector (6 μg) andheavy chain expression vector (4 μg) prepared using NucleoBond Xtra(Takara Bio Inc.) were added to 200 μL of Opti-Pro SFM (InvitrogenCorp.). 200 μL of the expression vector/Opti-Pro SFM mixed solution wasadded to 200 μL of the polyethyleneimine/Opti-Pro SFM mixed solution,and the mixture was gently stirred, further left for 5 minutes, and thenadded to the FreeStyle 293F cells. The cells were shake-cultured at 90rpm at 37° C. for 7 days in an 8% C02 incubator, and the obtainedculture supernatant was filtered through Minisart-Plus filter (SartoriusJapan K.K.) and used as a sample for evaluation.

Humanized hA2-11E-H3/L2 was obtained by the combination ofpCMA-G1/hA2-11E-H3 constructed in Example 18)-1-1 and pCMA/hA2-11E-L2constructed in Example 18)-2-1. Humanized hA2-11E-H3/L3 was obtained bythe combination of pCMA-G1/hA2-11E-H3 constructed in Example 18)-1-1 andpCMA/hA2-11E-L3 constructed in Example 18)-2-2. Humanized hA2-11E-H3/L4was obtained by the combination of pCMA-G1/hA2-11E-H3 constructed inExample 18)-1-1 and pCMA/hA2-11E-L4 constructed in Example 18)-2-3.Humanized hA2-11E-H4/L2 was obtained by the combination ofpCMA-G1/hA2-11E-H4 constructed in Example 18)-1-2 and pCMA/hA2-11E-L2constructed in Example 18)-2-1. Humanized hA2-11E-H4/L3 was obtained bythe combination of pCMA-G1/hA2-11E-H4 constructed in Example 18)-1-2 andpCMA/hA2-11E-L3 constructed in Example 18)-2-2. Humanized hA2-11E-H4/L4was obtained by the combination of pCMA-G1/hA2-11E-H4 constructed inExample 18)-1-2 and pCMA/hA2-11E-L4 constructed in Example 18)-2-3.Humanized hA2-25C-H3/L1 was obtained by the combination ofpCMA-G1/hA2-25C-H3 constructed in Example 18)-3-1 and pCMA-/hA2-25C-L1constructed in Example 18)-4-1. Humanized hA2-25C-H3/L2 was obtained bythe combination of pCMA-G1/hA2-25C-H3 constructed in Example 18)-3-1 andpCMA-/hA2-25C-L2 constructed in Example 18)-4-2. Humanized hA2-25C-H3/L3was obtained by the combination of pCMA-G1/hA2-25C-H3 constructed inExample 18)-3-1 and pCMA-/hA2-25C-L3 constructed in Example 18)-4-3.Humanized hA2-25C-H4/L1 was obtained by the combination ofpCMA-G1/hA2-25C-H4 constructed in Example 18)-3-2 and pCMA-/hA2-25C-L1constructed in Example 18)-4-1. Humanized hA2-25C-H4/L2 was obtained bythe combination of pCMA-G1/hA2-25C-H4 constructed in Example 18)-3-2 andpCMA-/hA2-25C-L2 constructed in Example 18)-4-2. Humanized hA2-25C-H4/L3was obtained by the combination of pCMA-G1/hA2-25C-H4 constructed inExample 18)-3-2 and pCMA-/hA2-25C-L3 constructed in Example 18)-4-3.

Example 19 Evaluation of In Vitro Activity of Humanized A2-11E Antibody(IgG1) and Humanized A2-25C Antibody (IgG1)

19)-1 Evaluation of the Antibodies in BMP-Induced OsteoblastDifferentiation Assay

The humanized A2-11E antibodies (IgG1) and the humanized A2-25Cantibodies (IgG1) prepared in Example 18)-5 were analyzed for theirinhibitory activity against intracellular signals through endogenousALK2, on the basis of their effects on BMP-induced osteoblastdifferentiation assay using C2C12 cells in the same way as in Example6)-2. 2.5 ng/mL GDF2/BMP9 (manufactured by R&D Systems, Inc.) was usedin the differentiation induction.

The results are shown in FIG. 48. The humanized A2-11E antibodies (IgG1)and the humanized A2-25C antibodies (IgG1) were confirmed to inhibit, ina dose-dependent manner, the differentiation of C2C12 cells intoosteoblast-like cells induced by BMP.

Example 20 Evaluation of Binding Activity of Humanized A2-11E Antibody(IgG1) and Humanized A2-25C Antibody (IgG1) Against Human ALK2

The dissociation constants of the humanized A2-11E antibodies (IgG1) andthe humanized A2-25C antibodies (IgG1) prepared in Example 18)-5 for theantigen (human ALK2 extracellular domain prepared in Example 14)-1) weremeasured using Biacore T200 (GE Healthcare Bio-Sciences Corp.) by thecapture method which involves capturing each antibody as a ligand ontoan immobilized anti-human IgG (Fc) antibody and assaying the antigen asan analyte. Approximately 1000 RU of the anti-human IgG (Fc) antibody(Human Antibody Capture kit, GE Healthcare Bio-Sciences Corp.) wascovalently bound to a sensor chip CM5 (GE Healthcare Bio-Sciences Corp.)by the amine coupling method. Similarly, this antibody was immobilizedonto a reference cell. The running buffer used was HBS-EP+(10 mM HEPES(pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.05% Surfactant P20). The culturesupernatant containing the antibody was added onto the anti-human IgG(Fc) antibody-immobilized chip for approximately 1 minute. Then,serially diluted solutions (0.78 to 200 nM) of the antigen were addedthereto at a flow rate of 30 μl/min for 300 seconds. Subsequently, thedissociation phase was monitored for 600 seconds. 3 M MgCl₂ was addedthereto as a regenerating solution at a flow rate of 10 μl/min for 30seconds. The data was analyzed using 1:1 binding models of analyticalsoftware (BIAevaluation software, version 1.0) to calculate anassociation rate constant ka, a dissociation rate constant kd, and adissociation constant (KD; KD=kd/ka).

The results are shown in Table 3.

TABLE 3 Dissociation constants of humanized A2-11E antibody andhumanized A2-25C antibody Name KD(nM) 1 hA2-11E-H3/L2 63.1 2hA2-11E-H3/L3 47.4 3 hA2-11E-H3/L4 31.3 4 hA2-11E-H4/L2 19.9 5hA2-11E-H4/L3 18.5 6 hA2-11E-H4/L4 11.1 7 hA2-25C-H3/L1 115.8 8hA2-25C-H3/L2 53.3 9 hA2-25C-H3/L3 64.4 10 hA2-25C-H4/L1 81.8 11hA2-25C-H4/L2 56.7 12 hA2-25C-H4/L3 27.9

Example 21 Epitope Analysis of A2-25C Antibody

21)-1 Preparation of Human Chimeric cA2-25C Fab Fragment

Human chimeric cA2-25C prepared in the same way as in Example 5)-8 wascleaved into Fab and Fc fragments using papain (Sigma-Aldrich Co. LLC),and these fragments were added to HiTrap Protein A HP column (GEHealthcare Bio-Sciences Corp.). The Fab fragment recovered as aflow-through fraction was concentrated.

21)-2 Crystallization and Structural Analysis of Human Chimeric cA2-25CFab Fragment and ALK2-ECD Complex

The protein complex of the chimeric A2-25C Fab fragment obtained inExample 21)-1 and the ALK2-ECD prepared according to Example 14 wereconcentrated to 3.8 mg/mL and used in crystallization trial employingvapor diffusion method. To 0.5 μL of the protein solution, an equalamount of a precipitant solution (0.15 M Li₂SO₄, 0.1 M Na citrate (pH3.4), 18% (w/v) PEG6,000, and 20% (v/v) ethylene glycol) was added, andthe resulting solution was placed in a sealed container containing 50 μLof a precipitant solution such that these solutions had no contact witheach other. The container was left standing at 20° C. Three days later,0.1 mm×0.05 mm×0.02 mm single crystals were obtained. The obtainedcrystals were frozen in liquid nitrogen. X-ray diffraction data wascollected under 95 K nitrogen stream using BL41XU of Spring8.Diffraction intensity was digitized from the obtained diffraction imageusing software mosflm (CCP4: Collaborative Computational Project No. 4)to determine crystal structure factors. The crystals were in theorthorhombic crystal system with a space group P2₁2₁2₁ and unit cells ofa=74.49 angstroms, b=128.05 angstroms, and c=147.73 angstroms. Themolecular replacement method was performed using the obtained structurefactors and the three-dimensional structure coordinates of Fab (antibodystructure determined by the past crystal structure analysis was used) todetermine the phases. Software phaser (CCP4: Collaborative ComputationalProject No. 4) was used in calculation. The crystal contained twocomplexes in the asymmetric unit. Structure refinement was performedusing software Refmac5 (CCP4), and model correction was performed usingsoftware coot. This operation was repetitively performed to obtain afinal R factor of 22.4% and a free R factor of 25.3% with a resolutionof 2.1 angstroms. The model consists of two complexes and contains aminoacid residues 1 to 212 of the A2-25C Fab L chain, amino acid residues 1to 219 of the A2-25C Fab H chain, amino acid residues 12 to 52 and 66 to88 of ALK2-ECD, and 411 water molecules. The determined amino acidresidues of ALK2-ECD commonly located within 4 angstroms from two A2-25CFabs are as follows: Glu18, Gly19, Leu20, Ile39, Asp41, Gly42, Phe43,His44, Val45, Tyr46, and Thr84. The ribbon model of the whole complex isshown in FIG. 49.

Example 22 Evaluation of Inhibitory Activity of A2-27D Against VariousALK2 Mutants

The inhibitory activity of A2-27 against 13 types of mutants (L196P,delP197_F198insL, R202I, R206H, Q207E, R258S, R258G, G325A, G328E,G328R, G328W, G356D, and R375P) identified so far from FOP cases, andwild-type ALK2 was analyzed in the same way as in Example 2)-3 usingHEK293A cells and BMP-specific Id1WT4F-luc luciferase reporter. 2.5hours after transfection, the medium was replaced with fresh OPTI-MEM I(manufactured by Life Technologies Corp.) containing 10 ng/mL BMP7(manufactured by Miltenyi Biotec) and 3 μg/mL rat IgG1 (manufactured byR&D Systems, Inc.) or A2-27D, and the cells were cultured overnight. Onthe next day, luciferase activity was measured using Dual-Glo LuciferaseAssay System (manufactured by Promega Corp.).

The results are shown in FIG. 50. The monoclonal antibody A2-27D wasconfirmed to inhibit luciferase activity induced by BMP7 in thewild-type ALK2 and all of the 13 types of mutants (FIG. 50).

INDUSTRIAL APPLICABILITY

The chimeric or humanized anti-ALK2 antibody of the present inventionhas an inhibitory effect on ALK2-mediated BMP signal transduction. Thepharmaceutical composition comprising the anti-ALK2 antibody can serveas a therapeutic or prophylactic drug for ectopic ossification and/orbone dysplasia, anemia, or diffuse intrinsic pontine glioma (DIPG).

FREE TEXT OF SEQUENCE LISTING

SEQ ID NO: 1: Nucleotide sequence of cDNA encoding the heavy chainvariable region of A2-11ESEQ ID NO: 2: Amino acid sequence of the heavy chain variable region ofA2-11ESEQ ID NO: 3: Nucleotide sequence of cDNA encoding the light chainvariable region of A2-11ESEQ ID NO: 4: Amino acid sequence of the light chain variable region ofA2-11ESEQ ID NO: 5: Nucleotide sequence of cDNA encoding the heavy chainvariable region of A2-15ASEQ ID NO: 6: Amino acid sequence of the heavy chain variable region ofA2-15ASEQ ID NO: 7: Nucleotide sequence of cDNA encoding the light chainvariable region of A2-15ASEQ ID NO: 8: Amino acid sequence of the light chain variable region ofA2-15ASEQ ID NO: 9: Nucleotide sequence of cDNA encoding the heavy chainvariable region of A2-25CSEQ ID NO: 10: Amino acid sequence of the heavy chain variable region ofA2-25CSEQ ID NO: 11: Nucleotide sequence of cDNA encoding the light chainvariable region of A2-25CSEQ ID NO: 12: Amino acid sequence of the light chain variable region ofA2-25CSEQ ID NO: 13: Nucleotide sequence of cDNA encoding the heavy chainvariable region of A2-27DSEQ ID NO: 14: Amino acid sequence of the heavy chain variable region ofA2-27DSEQ ID NO: 15: Nucleotide sequence of cDNA encoding the light chainvariable region of A2-27DSEQ ID NO: 16: Amino acid sequence of the light chain variable region ofA2-27DSEQ ID NO: 17: Nucleotide sequence of a DNA fragment containing asequence encoding the amino acids of a human κ chain secretory signaland a human κ chain constant regionSEQ ID NO: 18: Nucleotide sequence of a DNA fragment containing asequence encoding the amino acids of a human heavy chain secretorysignal and a human IgG1 constant regionSEQ ID NO: 19: Nucleotide sequence of the heavy chain of human chimericcA2-15ASEQ ID NO: 20: Amino acid sequence of the heavy chain of human chimericcA2-15ASEQ ID NO: 21: Nucleotide sequence of the light chain of human chimericcA2-15ASEQ ID NO: 22: Amino acid sequence of the light chain of human chimericcA2-15ASEQ ID NO: 23: Nucleotide sequence of the heavy chain of human chimericcA2-27DSEQ ID NO: 24: Amino acid sequence of the heavy chain of human chimericcA2-27DSEQ ID NO: 25: Nucleotide sequence of the light chain of human chimericcA2-27DSEQ ID NO: 26: Amino acid sequence of the light chain of human chimericcA2-27DSEQ ID NO: 27: Nucleotide sequence of humanized hA2-15A-H1SEQ ID NO: 28: Amino acid sequence of humanized hA2-15A-H1SEQ ID NO: 29: Nucleotide sequence of humanized hA2-15A-H4SEQ ID NO: 30: Amino acid sequence of humanized hA2-15A-H4SEQ ID NO: 31: Nucleotide sequence of a DNA fragment containing asequence encoding humanized hA2-15A-L1SEQ ID NO: 32: Amino acid sequence of humanized hA2-15A-L1SEQ ID NO: 33: Nucleotide sequence of a DNA fragment containing asequence encoding humanized hA2-15A-L4SEQ ID NO: 34: Amino acid sequence of humanized hA2-15A-L4SEQ ID NO: 35: Nucleotide sequence of a DNA fragment containing asequence encoding humanized hA2-15A-L6SEQ ID NO: 36: Amino acid sequence of humanized hA2-15A-L6SEQ ID NO: 37: Nucleotide sequence of a DNA fragment containing asequence encoding humanized hA2-15A-L7SEQ ID NO: 38: Amino acid sequence of humanized hA2-15A-L7SEQ ID NO: 39: Nucleotide sequence of humanized hA2-27D-H1SEQ ID NO: 40: Amino acid sequence of humanized hA2-27D-H1SEQ ID NO: 41: Nucleotide sequence of humanized hA2-27D-H2SEQ ID NO: 42: Amino acid sequence of humanized hA2-27D-H2SEQ ID NO: 43: Nucleotide sequence of humanized hA2-27D-H3SEQ ID NO: 44: Amino acid sequence of humanized hA2-27D-H3SEQ ID NO: 45: Nucleotide sequence of humanized hA2-27D-H4SEQ ID NO: 46: Amino acid sequence of humanized hA2-27D-H4SEQ ID NO: 47: Nucleotide sequence of humanized hA2-27D-H5SEQ ID NO: 48: Amino acid sequence of humanized hA2-27D-H5SEQ ID NO: 49: Nucleotide sequence of a DNA fragment containing asequence encoding humanized hA2-27D-L1SEQ ID NO: 50: Amino acid sequence of humanized hA2-27D-L1SEQ ID NO: 51: Nucleotide sequence of a DNA fragment containing asequence encoding humanized hA2-27D-L2SEQ ID NO: 52: Amino acid sequence of humanized hA2-27D-L2SEQ ID NO: 53: Nucleotide sequence of a DNA fragment containing asequence encoding humanized hA2-27D-L3SEQ ID NO: 54: Amino acid sequence of humanized hA2-27D-L3SEQ ID NO: 55: Nucleotide sequence of a DNA fragment containing asequence encoding humanized hA2-27D-L4SEQ ID NO: 56: Amino acid sequence of humanized hA2-27D-L4SEQ ID NO: 57: Nucleotide sequence of a DNA fragment containing asequence encoding humanized hA2-27D-L5SEQ ID NO: 58: Amino acid sequence of humanized hA2-27D-L5SEQ ID NO: 59: Amino acid sequence of A2-15A CDRH1SEQ ID NO: 60: Amino acid sequence of A2-15A CDRH2SEQ ID NO: 61: Amino acid sequence of A2-15A CDRH3SEQ ID NO: 62: Amino acid sequence of A2-15A CDRL1SEQ ID NO: 63: Amino acid sequence of A2-15A CDRL2SEQ ID NO: 64: Amino acid sequence of A2-15A CDRL3SEQ ID NO: 65: Amino acid sequence of A2-27D CDRH1SEQ ID NO: 66: Amino acid sequence of A2-27D CDRH2SEQ ID NO: 67: Amino acid sequence of A2-27D CDRH3SEQ ID NO: 68: Amino acid sequence of A2-27D CDRL1SEQ ID NO: 69: Amino acid sequence of A2-27D CDRL2SEQ ID NO: 70: Amino acid sequence of A2-27D CDRL3SEQ ID NO: 71: Amino acid sequence of humanized hA2-15A-L6 CDRL2SEQ ID NO: 72: Amino acid sequence of A2-11E CDRH1SEQ ID NO: 73: Amino acid sequence of A2-11E CDRH2SEQ ID NO: 74: Amino acid sequence of A2-11E CDRH3SEQ ID NO: 75: Amino acid sequence of A2-11E CDRL1SEQ ID NO: 76: Amino acid sequence of A2-11E CDRL2SEQ ID NO: 77: Amino acid sequence of A2-11E CDRL3SEQ ID NO: 78: Amino acid sequence of A2-25C CDRH1SEQ ID NO: 79: Amino acid sequence of A2-25C CDRH2SEQ ID NO: 80: Amino acid sequence of A2-25C CDRH3SEQ ID NO: 81: Amino acid sequence of A2-25C CDRL1SEQ ID NO: 82: Amino acid sequence of A2-25C CDRL2SEQ ID NO: 83: Amino acid sequence of A2-25C CDRL3SEQ ID NO: 84: Amino acid sequence of human ALK2SEQ ID NO: 85: Nucleotide sequence of human ALK2SEQ ID NO: 86: Amino acid sequence of mouse ALK2SEQ ID NO: 87: Nucleotide sequence of mouse ALK2

SEQ ID NOs: 88 to 102: Primer

SEQ ID NO: 103: Nucleotide sequence of a DNA fragment containing asequence encoding the amino acids of a human heavy chain signal sequenceand a human IgG2 constant regionSEQ ID NO: 104: Nucleotide sequence of humanized hA2-15A-H4 IgG2 typeSEQ ID NO: 105: Amino acid sequence of humanized hA2-15A-H4 IgG2 typeSEQ ID NO: 106: Nucleotide sequence of humanized hA2-27D-H2-LALASEQ ID NO: 107: Amino sequence of humanized hA2-27D-H2-LALASEQ ID NO: 108: Nucleotide sequence of humanized hA2-27D-H3-LALASEQ ID NO: 109: Amino sequence of humanized hA2-27D-H3-LALASEQ ID NO: 110: Nucleotide sequence of humanized hA2-11E-H3SEQ ID NO: 111: Amino acid sequence of humanized hA2-11E-H3SEQ ID NO: 112: Nucleotide sequence of humanized hA2-11E-H4SEQ ID NO: 113: Amino acid sequence of humanized hA2-11E-H4SEQ ID NO: 114: Nucleotide sequence of humanized hA2-11E-L2SEQ ID NO: 115: Amino acid sequence of humanized hA2-11E-L2SEQ ID NO: 116: Nucleotide sequence of humanized hA2-11E-L3SEQ ID NO: 117: Amino acid sequence of humanized hA2-11E-L3SEQ ID NO: 118: Nucleotide sequence of humanized hA2-11E-L4SEQ ID NO: 119: Amino acid sequence of humanized hA2-11E-L4SEQ ID NO: 120: Nucleotide sequence of humanized hA2-25C-H3SEQ ID NO: 121: Amino acid sequence of humanized hA2-25C-H3SEQ ID NO: 122: Nucleotide sequence of humanized hA2-25C-H4SEQ ID NO: 123: Amino acid sequence of humanized hA2-25C-H4SEQ ID NO: 124: Nucleotide sequence of humanized hA2-25C-L1SEQ ID NO: 125: Amino acid sequence of humanized hA2-25C-L1SEQ ID NO: 126: Nucleotide sequence of humanized hA2-25C-L2SEQ ID NO: 127: Amino acid sequence of humanized hA2-25C-L2SEQ ID NO: 128: Nucleotide sequence of humanized hA2-25C-L3SEQ ID NO: 129: Amino acid sequence of humanized hA2-25C-L3

SEQ ID NOs: 130 to 135: Primer

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. An antibody or an antigen-binding fragment thereof which inhibitsALK2-mediated BMP signal transduction and specifically binds to apolypeptide sequence consisting of at least 7 amino acids present in anyone of the following amino acid sequences (a) to (e): (a) the amino acidsequence of SEQ ID NO: 84; (b) an amino acid sequence consisting ofamino acid position numbers 21 to 123 of the amino acid sequence of SEQID NO: 84; (c) the amino acid sequence of SEQ ID NO: 86; (d) an aminoacid sequence consisting of amino acid position numbers 21 to 123 of theamino acid sequence of SEQ ID NO: 86; and (e) an amino acid sequencecomprising a substitution(s), a deletion(s), or an addition(s) of one toseveral amino acid residues in any one of the amino acid sequences (a)to (d), wherein the antibody or the antigen-binding fragment is amonoclonal antibody, a polyclonal antibody a chimeric antibody, ahumanized antibody, a human antibody, single-chain Fv, a bispecificantibody, or a multispecific antibody.
 2. A method for treating and/orpreventing ectopic ossification in a subject in need thereof, comprisingadministering to the subject an effective amount of claim 1, andoptionally at least one drug selected from the group consisting ofanti-inflammatory drugs, steroids, bisphosphonates, muscle relaxants,and retinoic acid receptor (RAR) γ agonists.
 3. The method according toclaim 2, wherein the ectopic ossification is fibrodysplasia ossificansprogressiva (FOP), progressive osseous heteroplasia (POH), traumaticectopic ossification, or ectopic ossification after implantarthroplasty.
 4. A method for treating and/or preventing a disease in asubject in need thereof, comprising administering to the subject aneffective amount of the antibody or antigen-binding fragment accordingto claim 1, wherein the disease is anemia or diffuse intrinsic pontineglioma (DIPG).
 5. A polynucleotide comprising (a) a polynucleotideselected from the group consisting of: a1) a nucleotide sequenceconsisting of nucleotide position numbers 58 to 426 of the nucleotidesequence of SEQ ID NO: 27; a2) a nucleotide sequence consisting ofnucleotide position numbers 58 to 426 of the nucleotide sequence of SEQID NO: 29; a3) a nucleotide sequence consisting of nucleotide positionnumbers 58 to 420 of the nucleotide sequence of SEQ ID NO: 39; a4) anucleotide sequence consisting of nucleotide position numbers 58 to 420of the nucleotide sequence of SEQ ID NO: 41; a5) a nucleotide sequenceconsisting of nucleotide position numbers 58 to 420 of the nucleotidesequence of SEQ ID NO: 43; a6) a nucleotide sequence consisting ofnucleotide position numbers 58 to 420 of the nucleotide sequence of SEQID NO: 45; a7) a nucleotide sequence consisting of nucleotide positionnumbers 58 to 420 of the nucleotide sequence of SEQ ID NO: 47; a8) anucleotide sequence consisting of nucleotide position numbers 58 to 411of the nucleotide sequence of SEQ ID NO: 110; a9) a nucleotide sequenceconsisting of nucleotide position numbers 58 to 411 of the nucleotidesequence of SEQ ID NO: 112; a10) a nucleotide sequence consisting ofnucleotide position numbers 58 to 411 of a nucleotide sequence of SEQ IDNO: 120; a11) a nucleotide sequence consisting of nucleotide positionnumbers 58 to 411 of a nucleotide sequence of SEQ ID NO: 122; a12) anucleotide sequence having at least 95% identity to any of thenucleotide sequences a1) to a11); a13) a nucleotide sequence having atleast 99% identity to any of the nucleotide sequences a1) to a11); a14)a nucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any of the nucleotide sequences a1) to a11);and a15) a nucleotide sequence comprising a substitution(s), adeletion(s), or an addition(s) of one to several nucleotides in any ofthe nucleotide sequences a1) to a11); and/or (b) a polynucleotideselected from the group consisting of: b1) a nucleotide sequenceconsisting of nucleotide position numbers 86 to 424 of the nucleotidesequence of SEQ ID NO: 31; b2) a nucleotide sequence consisting ofnucleotide position numbers 86 to 424 of the nucleotide sequence of SEQID NO: 33; b3) a nucleotide sequence consisting of nucleotide positionnumbers 86 to 424 of the nucleotide sequence of SEQ ID NO: 35; b4) anucleotide sequence consisting of nucleotide position numbers 86 to 424of the nucleotide sequence of SEQ ID NO: 37; b5) a nucleotide sequenceconsisting of nucleotide position numbers 86 to 412 of the nucleotidesequence of SEQ ID NO: 49; b6) a nucleotide sequence consisting ofnucleotide position numbers 86 to 412 of the nucleotide sequence of SEQID NO: 51; b7) a nucleotide sequence consisting of nucleotide positionnumbers 86 to 412 of the nucleotide sequence of SEQ ID NO: 53; b8) anucleotide sequence consisting of nucleotide position numbers 86 to 412of the nucleotide sequence of SEQ ID NO: 55; b9) a nucleotide sequenceconsisting of nucleotide position numbers 86 to 412 of the nucleotidesequence of SEQ ID NO: 57; b10) a nucleotide sequence consisting ofnucleotide position numbers 61 to 384 of the nucleotide sequence of SEQID NO: 114; b11) a nucleotide sequence consisting of nucleotide positionnumbers 61 to 384 of the nucleotide sequence of SEQ ID NO: 116; b12) anucleotide sequence consisting of nucleotide position numbers 61 to 384of the nucleotide sequence of SEQ ID NO: 118; b13) a nucleotide sequenceconsisting of nucleotide position numbers 61 to 387 of the nucleotidesequence of SEQ ID NO: 124; b14) a nucleotide sequence consisting ofnucleotide position numbers 61 to 387 of the nucleotide sequence of SEQID NO: 126; b15) a nucleotide sequence consisting of nucleotide positionnumbers 61 to 387 of the nucleotide sequence of SEQ ID NO: 128; b16) anucleotide sequence having at least 95% identity to any one nucleotidesequence selected from the nucleotide sequences b1) to b15); b17) anucleotide sequence having at least 99% identity to any one nucleotidesequence selected from the nucleotide sequences b1) to b15); b18) anucleotide sequence carried by a polynucleotide hybridizing understringent conditions to a polynucleotide consisting of a nucleotidesequence complementary to any one nucleotide sequence selected from thenucleotide sequences b1) to b15); and b19) a nucleotide sequencecomprising a substitution(s), a deletion(s), or an addition(s) of one toseveral nucleotides in any one nucleotide sequence selected from thenucleotide sequences b1) to b15).
 6. A vector comprising thepolynucleotide according to claim
 5. 7. A transformed host cellcomprising the polynucleotide according to claim
 5. 8. A transformedhost cell comprising a vector according to claim
 6. 9. The methodaccording to claim 2, wherein the antibody or antigen binding fragmentcomprises a heavy chain variable region having complementaritydetermining region (CDR)H1, CDRH2, and CDRH3 and a light chain variableregion having CDRL1, CDRL2, and CDRL3, wherein a set of the amino acidsequences of CDRH1, CDRH2, and CDRH3 and the amino acid sequences ofCDRL1, CDRL2, and CDRL3 is selected from the group consisting of: (1)SEQ ID NO: 72 for CDRH1, SEQ ID NO: 73 for CDRH2, and SEQ ID NO: 74 forCDRH3, and SEQ ID NO: 75 for CDRL1, SEQ ID NO: 76 for CDRL2, and SEQ IDNO: 77 for CDRL3; (2) SEQ ID NO: 59 for CDRH1, SEQ ID NO: 60 for CDRH2,and SEQ ID NO: 61 for CDRH3, and SEQ ID NO: 62 for CDRL1, SEQ ID NO: 63or 71 for CDRL2, and SEQ ID NO: 64 for CDRL3; (3) SEQ ID NO: 78 forCDRH1, SEQ ID NO: 79 for CDRH2, and SEQ ID NO: 80 for CDRH3, and SEQ IDNO: 81 for CDRL1, SEQ ID NO: 82 for CDRL2, and SEQ ID NO: 83 for CDRL3;and (4) SEQ ID NO: 65 for CDRH1, SEQ ID NO: 66 for CDRH2, and SEQ ID NO:67 for CDRH3, and SEQ ID NO: 68 for CDRL1, SEQ ID NO: 69 for CDRL2, andSEQ ID NO: 70 for CDRL3.
 10. The method according to claim 2, whereinthe antibody comprises a heavy chain variable region amino acid sequenceand a light chain variable region amino acid sequence of SEQ ID NO: 2and SEQ ID NO: 4; SEQ ID NO: 6 and SEQ ID NO: 8; SEQ ID NO: 6 and aminoacid residues 21 to 133 of SEQ ID NO: 36; SEQ ID NO: 10 and SEQ ID NO:12; or SEQ ID NO: 14 and SEQ ID NO: 16 respectively.
 11. The methodaccording to claim 2, wherein the antibody comprises (a) a heavy chainvariable region sequence selected from the group consisting of: a1) theamino acid sequence consisting of amino acid position numbers 20 to 142of the amino acid sequence of SEQ ID NO: 28; a2) the amino acid sequenceconsisting of amino acid position numbers 20 to 142 of the amino acidsequence of SEQ ID NO: 30 or SEQ ID NO: 105; a3) the amino acid sequenceconsisting of amino acid position numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 40; a4) the amino acid sequence consisting ofamino acid position numbers 20 to 140 of the amino acid sequence of SEQID NO: 42 or SEQ ID NO: 107; a5) the amino acid sequence consisting ofamino acid position numbers 20 to 140 of the amino acid sequence of SEQID NO: 44 or SEQ ID NO: 109; a6) the amino acid sequence consisting ofamino acid position numbers 20 to 140 of the amino acid sequence of SEQID NO: 46; a7) the amino acid sequence consisting of amino acid positionnumbers 20 to 140 of the amino acid sequence of SEQ ID NO: 48; a8) theamino acid sequence consisting of amino acid position numbers 20 to 137of the amino acid sequence of SEQ ID NO: 111; a9) the amino acidsequence consisting of amino acid position numbers 20 to 137 of theamino acid sequence of SEQ ID NO: 113; a10) the amino acid sequenceconsisting of amino acid position numbers 20 to 137 of the amino acidsequence of SEQ ID NO: 121; a11) the amino acid sequence consisting ofamino acid position numbers 20 to 137 of the amino acid sequence of SEQID NO: 123; a12) an amino acid sequence having at least 95% identity toany one amino acid sequence selected from the amino acid sequences a1)to a11); and a13) an amino acid sequence having at least 99% identity toany one amino acid sequence selected from the amino acid sequences a1)to a11); and (b) a light chain variable region sequence selected fromthe group consisting of: b1) the amino acid sequence consisting of aminoacid position numbers 21 to 133 of the amino acid sequence of SEQ ID NO:32; b2) the amino acid sequence consisting of amino acid positionnumbers 21 to 133 of the amino acid sequence of SEQ ID NO: 34; b3) theamino acid sequence consisting of amino acid position numbers 21 to 133of the amino acid sequence of SEQ ID NO: 36; b4) the amino acid sequenceconsisting of amino acid position numbers 21 to 133 of the amino acidsequence of SEQ ID NO: 38; b5) the amino acid sequence consisting ofamino acid position numbers 21 to 129 of the amino acid sequence of SEQID NO: 50; b6) the amino acid sequence consisting of amino acid positionnumbers 21 to 129 of the amino acid sequence of SEQ ID NO: 52; b7) theamino acid sequence consisting of amino acid position numbers 21 to 129of the amino acid sequence of SEQ ID NO: 54; b8) the amino acid sequenceconsisting of amino acid position numbers 21 to 129 of the amino acidsequence of SEQ ID NO: 56; b9) the amino acid sequence consisting ofamino acid position numbers 21 to 129 of the amino acid sequence of SEQID NO: 58; b10) the amino acid sequence consisting of amino acidposition numbers 21 to 128 of the amino acid sequence of SEQ ID NO: 115;b11) the amino acid sequence consisting of amino acid position numbers21 to 128 of the amino acid sequence of SEQ ID NO: 117; b12) the aminoacid sequence consisting of amino acid position numbers 21 to 128 of theamino acid sequence of SEQ ID NO: 119; b13) the amino acid sequenceconsisting of amino acid position numbers 21 to 129 of the amino acidsequence of SEQ ID NO: 125; b14) the amino acid sequence consisting ofamino acid position numbers 21 to 129 of the amino acid sequence of SEQID NO: 127; b15) the amino acid sequence consisting of amino acidposition numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 129;b16) an amino acid sequence having at least 95% identity to any oneamino acid sequence selected from the amino acid sequences b1) to b15);and b17) an amino acid sequence having at least 99% identity to any oneamino acid sequence selected from the amino acid sequences b1) to b15),wherein a combination of a heavy chain variable region sequence in (a)and a light chain variable region sequence in (b) that the monoclonalantibody comprises is selected from the group consisting of thecombinations of: a1) and b1); a1) and b2); a2) and b1); a2) and b2); a2)and b3); a2) and b4); a3) and b3); a4) and b5); a4) and b6); a4) andb7); a5) and b5); a5) and b6); a5) and b7); a6) and b5); a6) and b6);a6) and b7); a6) and b8); a7) and b7); a7) and b8); a7) and b9); a8) andb8); a9) and b6); a10) and b8); a11) and b10); a11) and b11); a11) andb12); a12) and b10); a12) and b11); a12) and b12); a13) and b13); a13)and b14); a13) and b15); a14) and b13); a14) and b14); and a14) andb15).
 12. The method according to claim 2, wherein the antibody is anantibody comprising (a) a heavy chain comprising the heavy chainvariable region sequence consisting of amino acid position numbers 20 to142 of the amino acid sequence of SEQ ID NO: 30 and a light chaincomprising the light chain variable region sequence consisting of aminoacid position numbers 21 to 133 of the amino acid sequence of SEQ ID NO:36; (b) a heavy chain comprising the amino acid sequence consisting ofamino acid position numbers 20 to 472 of the amino acid sequence of SEQID NO: 30 and a light chain comprising the amino acid sequenceconsisting of amino acid position numbers 21 to 238 of the amino acidsequence of SEQ ID NO: 36; (c) a heavy chain comprising the amino acidsequence consisting of amino acid position numbers 20 to 468 of theamino acid sequence of SEQ ID NO: 105 and a light chain comprising theamino acid sequence consisting of amino acid position numbers 21 to 238of the amino acid sequence of SEQ ID NO: 36; (d) a heavy chaincomprising the heavy chain variable region sequence consisting of aminoacid position numbers 20 to 140 of the amino acid sequence of SEQ ID NO:42 and a light chain comprising the light chain variable region sequenceconsisting of amino acid position numbers 21 to 129 of the amino acidsequence of SEQ ID NO: 52; (e) a heavy chain comprising the heavy chainvariable region sequence consisting of amino acid position numbers 20 to140 of the amino acid sequence of SEQ ID NO: 44 and a light chaincomprising the light chain variable region sequence consisting of aminoacid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO:56; (f) a heavy chain comprising the amino acid sequence consisting ofamino acid position numbers 20 to 470 of the amino acid sequence of SEQID NO: 42 and a light chain comprising the amino acid sequenceconsisting of amino acid position numbers 21 to 234 of the amino acidsequence of SEQ ID NO: 52; (g) a heavy chain comprising the amino acidsequence consisting of amino acid position numbers 20 to 470 of theamino acid sequence of SEQ ID NO: 44 and a light chain comprising theamino acid sequence consisting of amino acid position numbers 21 to 234of the amino acid sequence of SEQ ID NO: 56; (h) a heavy chaincomprising the amino acid sequence consisting of amino acid positionnumbers 20 to 470 of the amino acid sequence of SEQ ID NO: 107 and alight chain comprising the amino acid sequence consisting of amino acidposition numbers 21 to 234 of the amino acid sequence of SEQ ID NO: 52;or (i) a heavy chain comprising the amino acid sequence consisting ofamino acid position numbers 20 to 470 of the amino acid sequence of SEQID NO: 109 and a light chain comprising the amino acid sequenceconsisting of amino acid position numbers 21 to 234 of the amino acidsequence of SEQ ID NO:
 56. 13. The method according to claim 4, whereinthe antibody or antigen binding fragment comprises a heavy chainvariable region having complementarity determining region (CDR)H1,CDRH2, and CDRH3 and a light chain variable region having CDRL1, CDRL2,and CDRL3, wherein a set of the amino acid sequences of CDRH1, CDRH2,and CDRH3 and the amino acid sequences of CDRL1, CDRL2, and CDRL3 isselected from the group consisting of: (1) SEQ ID NO: 72 for CDRH1, SEQID NO: 73 for CDRH2, and SEQ ID NO: 74 for CDRH3, and SEQ ID NO: 75 forCDRL1, SEQ ID NO: 76 for CDRL2, and SEQ ID NO: 77 for CDRL3; (2) SEQ IDNO: 59 for CDRH1, SEQ ID NO: 60 for CDRH2, and SEQ ID NO: 61 for CDRH3,and SEQ ID NO: 62 for CDRL1, SEQ ID NO: 63 or 71 for CDRL2, and SEQ IDNO: 64 for CDRL3; (3) SEQ ID NO: 78 for CDRH1, SEQ ID NO: 79 for CDRH2,and SEQ ID NO: 80 for CDRH3, and SEQ ID NO: 81 for CDRL1, SEQ ID NO: 82for CDRL2, and SEQ ID NO: 83 for CDRL3; and (4) SEQ ID NO: 65 for CDRH1,SEQ ID NO: 66 for CDRH2, and SEQ ID NO: 67 for CDRH3, and SEQ ID NO: 68for CDRL1, SEQ ID NO: 69 for CDRL2, and SEQ ID NO: 70 for CDRL3.
 14. Themethod according to claim 4, wherein the antibody comprises a heavychain variable region amino acid sequence and a light chain variableregion amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 4; SEQ ID NO:6 and SEQ ID NO: 8; SEQ ID NO: 6 and amino acid residues 21 to 133 ofSEQ ID NO: 36; SEQ ID NO: 10 and SEQ ID NO: 12; or SEQ ID NO: 14 and SEQID NO: 16 respectively.
 15. The method according to claim 4, wherein theantibody comprises (a) a heavy chain variable region sequence selectedfrom the group consisting of: a1) the amino acid sequence consisting ofamino acid position numbers 20 to 142 of the amino acid sequence of SEQID NO: 28; a2) the amino acid sequence consisting of amino acid positionnumbers 20 to 142 of the amino acid sequence of SEQ ID NO: 30 or SEQ IDNO: 105; a3) the amino acid sequence consisting of amino acid positionnumbers 20 to 140 of the amino acid sequence of SEQ ID NO: 40; a4) theamino acid sequence consisting of amino acid position numbers 20 to 140of the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 107; a5) theamino acid sequence consisting of amino acid position numbers 20 to 140of the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 109; a6) theamino acid sequence consisting of amino acid position numbers 20 to 140of the amino acid sequence of SEQ ID NO: 46; a7) the amino acid sequenceconsisting of amino acid position numbers 20 to 140 of the amino acidsequence of SEQ ID NO: 48; a8) the amino acid sequence consisting ofamino acid position numbers 20 to 137 of the amino acid sequence of SEQID NO: 111; a9) the amino acid sequence consisting of amino acidposition numbers 20 to 137 of the amino acid sequence of SEQ ID NO: 113;a10) the amino acid sequence consisting of amino acid position numbers20 to 137 of the amino acid sequence of SEQ ID NO: 121; a11) the aminoacid sequence consisting of amino acid position numbers 20 to 137 of theamino acid sequence of SEQ ID NO: 123; a12) an amino acid sequencehaving at least 95% identity to any one amino acid sequence selectedfrom the amino acid sequences a1) to a11); and a13) an amino acidsequence having at least 99% identity to any one amino acid sequenceselected from the amino acid sequences a1) to a11); and (b) a lightchain variable region sequence selected from the group consisting of:b1) the amino acid sequence consisting of amino acid position numbers 21to 133 of the amino acid sequence of SEQ ID NO: 32; b2) the amino acidsequence consisting of amino acid position numbers 21 to 133 of theamino acid sequence of SEQ ID NO: 34; b3) the amino acid sequenceconsisting of amino acid position numbers 21 to 133 of the amino acidsequence of SEQ ID NO: 36; b4) the amino acid sequence consisting ofamino acid position numbers 21 to 133 of the amino acid sequence of SEQID NO: 38; b5) the amino acid sequence consisting of amino acid positionnumbers 21 to 129 of the amino acid sequence of SEQ ID NO: 50; b6) theamino acid sequence consisting of amino acid position numbers 21 to 129of the amino acid sequence of SEQ ID NO: 52; b7) the amino acid sequenceconsisting of amino acid position numbers 21 to 129 of the amino acidsequence of SEQ ID NO: 54; b8) the amino acid sequence consisting ofamino acid position numbers 21 to 129 of the amino acid sequence of SEQID NO: 56; b9) the amino acid sequence consisting of amino acid positionnumbers 21 to 129 of the amino acid sequence of SEQ ID NO: 58; b10) theamino acid sequence consisting of amino acid position numbers 21 to 128of the amino acid sequence of SEQ ID NO: 115; b11) the amino acidsequence consisting of amino acid position numbers 21 to 128 of theamino acid sequence of SEQ ID NO: 117; b12) the amino acid sequenceconsisting of amino acid position numbers 21 to 128 of the amino acidsequence of SEQ ID NO: 119; b13) the amino acid sequence consisting ofamino acid position numbers 21 to 129 of the amino acid sequence of SEQID NO: 125; b14) the amino acid sequence consisting of amino acidposition numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 127;b15) the amino acid sequence consisting of amino acid position numbers21 to 129 of the amino acid sequence of SEQ ID NO: 129; b16) an aminoacid sequence having at least 95% identity to any one amino acidsequence selected from the amino acid sequences b1) to b15); and b17) anamino acid sequence having at least 99% identity to any one amino acidsequence selected from the amino acid sequences b1) to b15), wherein acombination of a heavy chain variable region sequence in (a) and a lightchain variable region sequence in (b) that the monoclonal antibodycomprises is selected from the group consisting of the combinations of:a1) and b1); a1) and b2); a2) and b1); a2) and b2); a2) and b3); a2) andb4); a3) and b3); a4) and b5); a4) and b6); a4) and b7); a5) and b5);a5) and b6); a5) and b7); a6) and b5); a6) and b6); a6) and b7); a6) andb8); a7) and b7); a7) and b8); a7) and b9); a8) and b8); a9) and b6);a10) and b8); a11) and b10); a11) and b11); a11) and b12); a12) andb10); a12) and b11); a12) and b12); a13) and b13); a13) and b14); a13)and b15); a14) and b13); a14) and b14); and a14) and b15).
 16. Themethod according to claim 4, wherein the antibody is an antibodycomprising (a) a heavy chain comprising the heavy chain variable regionsequence consisting of amino acid position numbers 20 to 142 of theamino acid sequence of SEQ ID NO: 30 and a light chain comprising thelight chain variable region sequence consisting of amino acid positionnumbers 21 to 133 of the amino acid sequence of SEQ ID NO: 36; (b) aheavy chain comprising the amino acid sequence consisting of amino acidposition numbers 20 to 472 of the amino acid sequence of SEQ ID NO: 30and a light chain comprising the amino acid sequence consisting of aminoacid position numbers 21 to 238 of the amino acid sequence of SEQ ID NO:36; (c) a heavy chain comprising the amino acid sequence consisting ofamino acid position numbers 20 to 468 of the amino acid sequence of SEQID NO: 105 and a light chain comprising the amino acid sequenceconsisting of amino acid position numbers 21 to 238 of the amino acidsequence of SEQ ID NO: 36; (d) a heavy chain comprising the heavy chainvariable region sequence consisting of amino acid position numbers 20 to140 of the amino acid sequence of SEQ ID NO: 42 and a light chaincomprising the light chain variable region sequence consisting of aminoacid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO:52; (e) a heavy chain comprising the heavy chain variable regionsequence consisting of amino acid position numbers 20 to 140 of theamino acid sequence of SEQ ID NO: 44 and a light chain comprising thelight chain variable region sequence consisting of amino acid positionnumbers 21 to 129 of the amino acid sequence of SEQ ID NO: 56; (f) aheavy chain comprising the amino acid sequence consisting of amino acidposition numbers 20 to 470 of the amino acid sequence of SEQ ID NO: 42and a light chain comprising the amino acid sequence consisting of aminoacid position numbers 21 to 234 of the amino acid sequence of SEQ ID NO:52; (g) a heavy chain comprising the amino acid sequence consisting ofamino acid position numbers 20 to 470 of the amino acid sequence of SEQID NO: 44 and a light chain comprising the amino acid sequenceconsisting of amino acid position numbers 21 to 234 of the amino acidsequence of SEQ ID NO: 56; (h) a heavy chain comprising the amino acidsequence consisting of amino acid position numbers 20 to 470 of theamino acid sequence of SEQ ID NO: 107 and a light chain comprising theamino acid sequence consisting of amino acid position numbers 21 to 234of the amino acid sequence of SEQ ID NO: 52; or (i) a heavy chaincomprising the amino acid sequence consisting of amino acid positionnumbers 20 to 470 of the amino acid sequence of SEQ ID NO: 109 and alight chain comprising the amino acid sequence consisting of amino acidposition numbers 21 to 234 of the amino acid sequence of SEQ ID NO: 56.